Pharmaceutical combinations for the treatment of cancer

ABSTRACT

The disclosure herein provides combination therapies for the treatment of cancers such as Leukemia, lymphoma and triple negative breast cancer. The disclosure provides combination therapies of CDK inhibitors, e.g., a CDK inhibitor represented by Formula (I): or a pharmaceutically acceptable salt thereof together with a BCL-2 inhibitor or proteasome inhibitor for the treatment of cancer.

CROSS-REFERENCE

This application claims priority to U.S. Provisional Patent ApplicationSer. No. 62/314,356, filed Mar. 28, 2016, the entire contents of whichare incorporated herein by reference.

BACKGROUND

Numerous cancer-related therapeutics are under phase I or phase IIclinical trial and evaluations at any particular time; however, most ofthem will fail to advance. In fact, it is estimated that more than 90%of cancer-related therapeutics will fail phase I or II clinical trialevaluation. The failure rate in phase III trials is almost 50%, and thecost of new drug development from discovery through phase III trials isbetween $0.8 billion and $1.7 billion and can take between eight and tenyears.

In addition, many patients fail to respond even to standard drugs thathave been shown to be efficacious. For reasons that are not currentlywell understood or easily evaluated, individual patients may not respondto standard drug therapy. In some cases, administration of drugcombinations may be more efficacious for treating cancer than drugsadministered individually. These drug combinations may actsynergistically to enhance the anti-cancer activity of the drugs. Insome cases, drugs that are not particularly efficacious may find new andunexpected uses when combined with additional drug therapies.

SUMMARY

In one aspect, the disclosure provides a method of treating a bloodcancer comprising administering to a subject in need thereof atherapeutically effective amount of a CDK inhibitor represented byFormula I:

or a pharmaceutically acceptable salt thereof, wherein:

-   -   R₁ is optionally substituted phenyl;    -   R₂ and R₃ are each independently selected from hydroxy and OR₈,        wherein R₈ is optionally substituted C₁-C₁₀-alkyl;    -   R₄ is optionally substituted C₁-C₄-alkyl; and    -   R₉ is hydrogen or optionally substituted C₁-C₄-alkyl;        and a therapeutically effective amount of a BCL-2 inhibitor.

In certain aspects, the disclosure provides a method of treating acancer comprising administering to a subject in need thereof atherapeutically effective amount of a CDK inhibitor represented byFormula I:

or a pharmaceutically acceptable salt thereof, wherein:

-   -   R₁ is optionally substituted phenyl;    -   R₂ and R₃ are each independently selected from hydroxy and OR₈,        wherein R₈ is optionally substituted C₁-C₁₀-alkyl;    -   R₄ is optionally substituted C₁-C₄-alkyl; and    -   R₉ is hydrogen or optionally substituted C₁-C₄-alkyl;        and a therapeutically effective amount of a proteasome        inhibitor. In certain embodiments, the cancer is selected from a        blood cancer and triple negative breast cancer (TNBC).

In certain embodiments, the compound of Formula I is represented byFormula Ia:

or a pharmaceutically acceptable salt thereof.

In certain embodiments for a compound or salt of Formula I or Ia, R₁ isoptionally substituted with one or more substituents independentlyselected from hydroxy, cyano, halo, amino, C₁-C₄-alkyl, C₁-C₄-alkoxy,C₁-C₄-hydroxyalkyl, C₁-C₄-haloalkyl, and nitro. In certain embodiments,R₁ is substituted with one or more substituents independently selectedfrom halo and C₁-C₄-haloalkyl. In certain embodiments, R₁ is2-chloro-4-trifluoromethylphenyl.

In certain embodiments for a compound or salt of Formula I or Ia, R₂ andR₃ are each independently selected from hydroxy and OR₈, wherein R₈ isC₁-C₁₀-alkyl optionally substituted with one or more substituentsindependently selected from hydroxy, cyano, halo, amino, ═O, ═S,C₁-C₄-alkoxy, and nitro. In certain embodiments, R₂ and R₃ are eachhydroxy.

In certain embodiments for a compound or salt of Formula I or Ia, R₄ isC₁-C₄-alkyl substituted with one or more substituents selected fromhydroxy, cyano, halo, amino, ═O, ═S, C₁-C₄-alkoxy, and nitro. In certainembodiments, R₄ is C₁-C₄-alkyl substituted with one or more substituentsselected from hydroxy, cyano, halo, amino, ═O, ═S, C₁-C₄-alkoxy, andnitro. In certain embodiments, R₄ is 2-hydroxymethyl.

In certain embodiments for a compound or salt of Formula I or Ia, R₉ isC₁-C₄-alkyl optionally substituted with hydroxy, cyano, halo, amino, ═O,═S, C₁-C₄-alkoxy, and nitro. In certain embodiments, R₉ is methyl. Incertain embodiments the compound of Formula I is represented by formulaIb:

or a pharmaceutically acceptable salt thereof.

In certain embodiments, the BCL-2 inhibitor of the methods describedherein is a BH3-mimetic. The BCL-2 inhibitor may specifically inhibitthe Bcl-2 protein. The BCL-2 inhibitor may be selected from navitoclax,venetoclax, A-1155463, A-1331852, ABT-737, obatoclax, S44563, TW-37,A-1210477, AT101, HA14-1, BAM7, sabutoclax, UMI-77, gambogic acid,maritoclax, MIM1, methylprednisolone, iMAC2, Bax inhibitor peptide V5,Bax inhibitor peptide P5, Bax channel blocker, and ARRY 520trifluoroacetate. In certain embodiments, the BCL-2 inhibitor of themethods described herein is selected from navitoclax and venetoclax or apharmaceutically acceptable salt of either one thereof.

In certain embodiments, the blood cancer of the methods described hereinis selected from acute myeloid leukemia (AML), chronic myeloid leukemia(CML), acute lymphocytic lymphoma (ALL), and chronic lymphocyticleukemia (CLL), diffuse large B-cell lymphoma (DLBCL), primarymediastinal B-cell lymphoma, intravascular large B-cell lymphoma,follicular lymphoma, small lymphocytic lymphomia (SLL), mantle celllymphoma, marginal zone B-cell lymphomas, extranodal marginal zoneB-cell lymphomas, nodal marginal zone B-cell lymphoma, splenic marginalzone B-cell lymphoma, Burkitt lymphoma, lymphoplasmacytic lymphoma, andprimary central nervous system lymphoma. The blood cancer may be diffuselarge B-cell lymphoma, acute myeloid leukemia or chronic lymphocyticleukemia.

For certain methods described herein, the CDK inhibitor and BCL-2inhibitor may be administered concurrently. For the methods describedherein, the CDK inhibitor and BCL-2 inhibitor may be administeredsequentially within about 12 hours of each other, such as within about 5hours of each other.

For certain methods described herein, the CDK inhibitor and BCL-2inhibitor may be co-formulated in a pharmaceutical composition.

For certain methods described herein, the CDK inhibitor and BCL-2inhibitor may be administered daily, every other day or every third day.

For certain methods described herein, the proteasome inhibitor isselected from bortezomib, marizomib, ixazomib, disulfiram,epigallocatechin-3-gallate, salinosporamide A, carfilzomib, ONX 0912,CEP-18770, MLN9708, epoxomicin, MG132 and a pharmaceutically acceptablesalt of any one thereof. In certain embodiments, the proteasomeinhibitor is selected from bortezomib, marizomib, ixazomib, and apharmaceutically acceptable salt of any one thereof.

In certain methods described herein, the CDK inhibitor and proteasomeinhibitor are administered concurrently. The CDK inhibitor andproteasome inhibitor may be administered sequentially within about 12hours of each other, such as within 5 hours of each other.

In certain methods described herein, the CDK inhibitor and proteasomeinhibitor are co-formulated in a pharmaceutical composition.

In certain methods described herein, the CDK inhibitor and BCL-2inhibitor are administered daily, every other day or every third day.

In certain aspects the disclosure provides a pharmaceutical compositioncomprising a therapeutically effective amount of a CDK inhibitorrepresented by Formula I:

or a pharmaceutically acceptable salt thereof, wherein:

-   -   R₁ is optionally substituted phenyl;    -   R₂ and R₃ are each independently selected from hydroxy and OR₈,        wherein R₈ is optionally substituted C₁-C₁₀-alkyl;    -   R₄ is optionally substituted C₁-C₄-alkyl; and    -   R₉ is hydrogen or optionally substituted C₁-C₄-alkyl;        a therapeutically effective amount of a BCL-2 inhibitor or a        proteasome inhibitor, and a pharmaceutically acceptable        excipient.

In certain embodiments, the compound or salt of Formula I is representedby Formula Ia:

For the compositions described herein, for a compound or salt of FormulaI or Ia, R₁ may be optionally substituted with one or more substituentsindependently selected from hydroxy, cyano, halo, amino, C₁-C₄-alkyl,C₁-C₄-alkoxy, C₁-C₄-hydroxyalkyl, C₁-C₄-haloalkyl, and nitro. In certainembodiments, R₁ is substituted with one or more substituentsindependently selected from halo and C₁-C₄-haloalkyl. In certainembodiments, R₁ is 2-chloro-4-trifluoromethylphenyl.

For the compositions described herein, for a compound or salt of FormulaI or Ia, R₂ and R₃ may each independently selected from hydroxy and OR₈,wherein R₈ is C₁-C₁₀-alkyl optionally substituted with one or moresubstituents independently selected from hydroxy, cyano, halo, amino,═O, ═S, C₁-C₄-alkoxy, and nitro. In certain embodiments, R₂ and R₃ areeach hydroxy.

For the compositions described herein, for a compound or salt of FormulaI or Ia, R₄ is C₁-C₄-alkyl substituted with one or more substituentsselected from hydroxy, cyano, halo, amino, ═O, ═S, C₁-C₄-alkoxy, andnitro. In certain embodiments, R₄ is C₁-C₄-alkyl substituted with one ormore substituents selected from hydroxy, cyano, halo, amino, ═O, ═S,C₁-C₄-alkoxy, and nitro. In certain embodiments, R₄ is 2-hydroxymethyl.

For the compositions described herein, for a compound or salt of FormulaI or Ia, R₉ may be C₁-C₄-alkyl optionally substituted with hydroxy,cyano, halo, amino, ═O, ═S, C₁-C₄-alkoxy, and nitro. In certainembodiments, R₉ is methyl.

For the compositions described herein, the compound of Formula I may berepresented by Formula Ib:

or a pharmaceutically acceptable salt thereof.

For the compositions described herein comprising a BCL-2 inhibitor, theBCL-2 inhibitor may be selected from navitoclax, venetoclax, A-1155463,A-1331852, ABT-737, obatoclax, S44563, TW-37, A-1210477, AT101, HA14-1,BAM7, sabutoclax, UMI-77, gambogic acid, maritoclax, MIM1,methylprednisolone, iMAC2, Bax inhibitor peptide V5, Bax inhibitorpeptide P5, Bax channel blocker, ARRY 520 trifluoroacetate and apharmaceutically acceptable salt of any one thereof. The BCL-2 inhibitormay be selected from navitoclax and venetoclax or a pharmaceuticallyacceptable salt of either one thereof. In certain embodiments, the BCL-2inhibitor is venetoclax.

For the compositions described herein comprising a proteasome inhibitor,the proteasome inhibitor may be selected from bortezomib, marizomib,ixazomib, disulfiram, epigallocatechin-3-gallate, salinosporamide A,carfilzomib, ONX 0912, CEP-18770, MLN9708, epoxomicin, MG132 and apharmaceutically acceptable salt of any one thereof. In certainembodiments, the proteasome inhibitor is selected from bortezomib,marizomib, ixazomib, and a pharmaceutically acceptable salt of any onethereof.

INCORPORATION BY REFERENCE

All publications, patents, and patent applications mentioned in thisspecification are herein incorporated by reference to the same extent asif each individual publication, patent, or patent application wasspecifically and individually indicated to be incorporated by reference.

BRIEF DESCRIPTION OF THE DRAWINGS

The novel features of the invention are set forth with particularity inthe appended claims. A better understanding of the features andadvantages of the present invention will be obtained by reference to thefollowing detailed description that sets forth illustrative embodiments,in which the principles of the invention are utilized, and theaccompanying drawings of which:

FIG. 1 illustrates a comparison of voruciclib and flavopiradol activityagainst 38 kinases.

FIG. 2 illustrates single-digit nM potency of voruciclib againstcyclin-dependent kinases.

FIGS. 3A-3D illustrate a synergistic effect of voruciclib in combinationwith venetoclax (ABT-199). Voruciclib inhibits the induction of inducedmyeloid leukemia cell differentiation protein (MLC-1) by venetoclax inNU-DHL-1 diffuse large B-cell lymphoma (DLBCL) cells. MCL-1=red;DAPI=blue; fluorescent tracking marker (FTM)=green.

FIGS. 4A-4D illustrate increased apoptosis in NU-DHL-1 diffuse largeB-cell lymphoma (DLBCL) cells by combination treatment with vorucicliband venetoclax. Cleaved caspase-3 (CC3)=red; DAPI=blue; fluorescenttracking marker (FTM)=green.

FIGS. 5A-5D illustrate a synergistic effect of voruciclib in combinationwith navitoclax (ABT-263). Treatment of the Ramos Burkitt's lymphomacell line with voruciclib and navitoclax induces apoptosis. Cleavedcaspase-3 (CC3)=red; DAPI=blue; fluorescent tracking marker (FTM)=green.

FIGS. 6A-6E illustrate a synergistic effect of voruciclib in combinationwith venetoclax across five models of diffuse large B-cell lymphoma.

FIG. 7 illustrates that inhibition of MCL-1 through CDK9 helps shiftcells to apoptosis.

FIGS. 8A-8D illustrates that proteasome inhibition induces upregulationof MCL-1 in triple negative breast cancer (TNBC). FIG. 8A illustrates alist of compounds screened in HCC1187 TNBC xenograft model. FIG. 8Billustrates the mean % change in MCL-1 induction by treatment withvarious compounds. FIGS. 8C-8D illustrate staining of cells for CC3(shown in red) with vehicle and after treatment with bortezomib.

FIGS. 9A-9D illustrate a synergistic effect of voruciclib in combinationwith marizomib on NudHL1 DLBCL cells. Cleaved caspase-3 (CC3)=red;DAPI=blue; fluorescent tracking marker (FTM)=green.

FIGS. 10A-10D illustrate a synergistic effect of voruciclib incombination with bortezomib on NudHL1 DLBCL cells. Cleaved caspase-3(CC3)=red; DAPI=blue; fluorescent tracking marker (FTM)=green.

FIGS. 11A-11D illustrate a synergistic effect of voruciclib incombination with bortezomib on triple-negative breast cancer cells.Cleaved caspase-3 (CC3)=red; DAPI=blue; fluorescent tracking marker(FTM)=green.

FIGS. 12A-12E illustrate a synergistic effect of voruciclib incombination with bortezomib on HCC1187 triple-negative breast cancercells. FIGS. 12A-12D illustrate decreased tumor volume in an HCC1187TNBC mouse model treated with voruciclib and bortezomib.

FIG. 12E illustrates a Western blot demonstrating decreased MCL-1expression in HCC1187 TNBC cells treated with voruciclib and bortezomibor voruciclib and tunicamycin.

FIG. 13 illustrates that effect of voruciclid and bortezomib and thecombination of voruciclib and bortezomib on body weight in an HCC1187TNBC mouse model.

FIG. 14 illustrates the effect of bortezomib in combination withpalbociclib, a CDK4/6 inhibitor, in an HCC1187 TNBC mouse model.

FIGS. 15A-15B illustrate that voruciclib diminishes bortezomib-inducedMCL-1 and E3 ubiquitin-protein ligase XIAP expression. FIG. 15Aillustrates a proposed model of voruciclib inhibition of CDK9. FIG. 15Billustrates a Western blot demonstrating that voruciclib diminishesbortezomib-induced increase in MCL-1 and E3 ubiquitin-protein ligaseXIAP expression.

FIGS. 16A-16B illustrates cells resistant to bortezomib treatment. FIG.16A illustrates cells resistant to bortezomib treatment in an areaotherwise cleared of cells by bortezomib treatment. FIG. 16B illustratesthat cells resistant to bortezomib express GRP78, a protein expressed aspart of the ER stress response.

FIG. 17 illustrates three different ER stress response pathways.

FIGS. 18A-18B illustrate that voruciclib may affect the IRE1α-dependentER stress response pathway. FIG. 18A illustrates the IRE1α-dependent ERstress response pathway. FIG. 18B illustrates a Western blotdemonstrating that the ER stress inducer tunicamycin dramaticallyupregulates X-box binding protein 1 (XBP1), a pro-survival (anti-tumorcell death) protein. This effect is dramatically mitigated byvoruciclib. At 6 hours, only tunicamycin illustrates this effect, but at24 hours, both bortezomib and tunicamycin illustrate this effect.

FIGS. 19A-19B illustrate repression of bortezomib-induced XBP1transcription by voruciclib. STF083010=IRE1α endoribonuclease activityinhibitor; Tm=Tunicamycin

FIGS. 20A-20D illustrate a synergistic effect of voruciclib incombination with ixazomib. Cleaved-caspase 3 (CC3)=red; DAPI=blue;fluorescent tracking marker (FTM)=green.

FIGS. 21A-21B illustrate a solid tumor section treated with CDKinhibitors, voruciclib, palbociclib, dinaciclib and flavopiradol incombination with ixazomib. Only Voruciclib+Ixazomib reproducibly lead toovert tumor cell clearing within 24 hr.

FIGS. 22A-B illustrates the synergy from voruciclib and venetoclax inthe SU-DHL-4 model of diffuse large B-cell lymphoma (DLBCL).

FIGS. 23A-23C illustrate the synergy from voruciclib and venetoclax inthe SU-DHL-4 model, the OCI Ly10 model, and the U2932 model of diffuselarge B-cell lymphoma (DLBCL).

FIG. 24 illustrates that voruciclib suppresses MCL1 expression in DLBCLxenograft tumors.

FIGS. 25A-25B illustrate the synergistic effect of voruciclib andvenetoclax in ABC-type (RIVA) DLBCL in mice.

FIGS. 26A-26B illustrate the synergistic effect of voruciclib andvenetoclax and the effect on body weight in U2932 model if DLBCL inmice.

FIGS. 27A-27B illustrate the synergistic effect of voruciclib andvenetoclax in NUDHL1 model of DLBCL in mice.

FIG. 28 illustrates the synergistic effect of voruciclib and venetoclaxin SUDHL4 model of GC DLBCL.

FIG. 29 illustrates that voruciclib restores p53 abrogated byvenetoclax.

FIGS. 30A-30C illustrate that voruciclib has single agent activity inAML cell lines.

FIG. 31 illustrates that the combination of voruciclib and venetoclaxinduce synergistic cell death in AML cell lines.

FIG. 32 illustrates the synergistic effect that the combination ofvoruciclib and venetoclax impedes tumor growth in SKM1 AML xenografts.

FIG. 33 illustrates that voruciclib-induced apoptosis correlates withrepression of MCL.

DETAILED DESCRIPTION

The disclosure provides combination therapies for the treatment ofcancer. In particular, the disclosure provides combination therapies ofCDK inhibitors with other anticancer agents for treating cancer. In oneaspect the disclosure provides compositions and methods for treatingcancer with a CDK inhibitor in combination with a BCL-2 inhibitor. Suchcombination provides synergistic effects in the treatment of cancers andparticularly treatment of blood cancers, e.g., leukemia and lymphoma.

In another aspect, the disclosure provides compositions and methods fortreating cancer with a CDK inhibitor in combination with a proteasomeinhibitor. Such combination provides a synergistic effect in thetreatment of cancer and particularly treatment of blood cancers andtriple negative breast cancer.

The general terms used hereinbefore and hereinafter preferably have thefollowing meanings within the context of this disclosure, unlessotherwise indicated. Thus, the definitions of the general terms as usedin the context of the present invention are provided herein below:

The singular forms “a,” “an,” and “the” include plural reference unlessthe context clearly dictates otherwise.

The term “about,” as used herein, generally refers to an acceptableerror range for the particular value as determined by one of ordinaryskill in the art, which may depend in part on how the value is measuredor determined. For example, “about” can mean within 1 or more than 1standard deviation. Alternatively, “about” can mean a range of up to20%, up to 10%, up to 5%, or up to 1% of a given value. Alternatively,particularly with respect to biological systems or processes, the termcan mean within an order of magnitude, within 5-fold, and within 2-fold,of a value.

As used herein, the term “at least one” is refers to one or more. Forinstance, the term “at least one anticancer agent” means that thecombination comprises a single anticancer agent or more anticanceragents.

The term “effective amount” or “therapeutically effective amount,” asused herein, generally refers to an amount of a compound describedherein that is sufficient to affect an intended, predetermined orprescribed application, including but not limited to, disease orcondition treatment. The therapeutically effective amount can varydepending upon the application (e.g., in vitro or in vivo), or thesubject and disease condition being treated, e.g., the weight and age ofthe subject, the severity of the disease condition and the manner ofadministration. The term also may apply to a dose that induces aparticular response in target cells, e.g., reduction of proliferation ordown regulation of activity of a target protein. The specific dose mayvary depending on the particular compounds chosen, the dosing regimen tobe followed, whether it is administered in combination with othercompounds, timing of administration, the tissue to which it isadministered, and the physical delivery system in which it is carried.

As used herein, the term “pharmaceutically acceptable” means that thecarrier, diluent, excipients, and/or salt must be compatible with theother ingredients of the formulation, and not deleterious to therecipient thereof “Pharmaceutically acceptable” also means that thecompositions or dosage forms are within the scope of sound medicaljudgment, suitable for use for an animal or human without excessivetoxicity, irritation, allergic response, or other problem orcomplication, commensurate with a reasonable benefit/risk ratio.

As used herein, the term “combination” or “pharmaceutical combination”refers to the combined administration of the anticancer agents.Combinations of the disclosure include a CDK inhibitor, e.g., a compoundof Formula I, Ia, or Ib, and at least one anticancer agent selected froma BCL-2 inhibitor and a proteasome inhibitor; which anti-cancer agentsmay be administered to a subject in need thereof, e.g., concurrently orsequentially.

The term “synergistic,” or “synergistic effect” or “synergism” as usedherein, generally refers to an effect such that the one or more effectsof the combination of compositions is greater than the one or moreeffects of each component alone, or they can be greater than the sum ofthe one or more effects of each component alone. The synergistic effectcan be greater than about 10%, 20%, 30%, 50%, 75%, 100%, 110%, 120%,150%, 200%, 250%, 350%, or 500% or more than the effect on a subjectwith one of the components alone, or the additive effects of each of thecomponents when administered individually. The effect can be any of themeasurable effects described herein. Advantageously, such synergybetween the agents when combined, may allow for the use of smaller dosesof one or both agents, may provide greater efficacy at the same doses,and may prevent or delay the build-up of multi-drug resistance. Thecombination index (CI) method of Chou and Talalay may be used todetermine the synergy, additive or antagonism effect of the agents usedin combination. When the CI value is less than 1, there is synergybetween the compounds used in the combination; when the CI value isequal to 1, there is an additive effect between the compounds used inthe combination and when CI value is more than 1, there is anantagonistic effect. The synergistic effect may be attained byco-formulating the agents of the pharmaceutical combination. Thesynergistic effect may be attained by administering two or more agentsas separate formulations administered simultaneously or sequentially.

Cyclin-dependent kinases (CDKs) are a family of enzymes which becomeactivated in specific phases of the cell cycle. CDKs consist of acatalytic subunit (the actual cyclin-dependent kinase or CDK) and aregulatory subunit (cyclin). There are at least nine CDKs (CDK1, CDK2,CDK3, CDK4, CDK5, CDK6, CDK7, CDK8, CDK9, etc.) and at least 15different types of cyclins (cyclin A, B1, B2, D1, D2, D3, E, H etc.).Each step of the cell cycle is regulated by such CDK complexes: G1/Stransition (CDK2/cyclin A, CDK4/cyclin D1-D3, CDK6/cyclin D3), S phase(CDK2/cyclin A), G2 phase 30 (CDK1/cyclin A), G2/M transition phase(CDK1/cyclin B).

As used herein, the term “CDK inhibitor” refers to an agent that iscapable of inhibiting one or more cyclin dependent kinases (CDK).Aberrant expression and overexpression of these kinases are evidenced inmany disease conditions such as cancer. In the context of the presentinvention, the CDK inhibitor of the pharmaceutical combination describedherein may be a compound of Formula I, Ia, or Ib or a pharmaceuticallyacceptable salt thereof. The compounds of the present disclosure mayinhibit one or more of CDK1/cyclin B, CDK2/cyclin E, CDK4/cyclin D,CDK4/cyclin D1 and CDK9/cyclin T1 with specificity. In certainembodiments, a compound of the disclosure inhibits CDK9/cyclin T1 orCDK9 with specificity.

Disclosed herein are combination therapies for the treatment of cancer,e.g., leukemia, lymphoma and breast cancer. The methods and compositionsdescribed herein may include a cyclin-dependent kinase (CDK) inhibitor,such as a compound of Formula I, Ia, or Ib or a pharmaceuticallyacceptable salt thereof. In some cases, a combination therapy mayinclude a CDK inhibitor in combination with a proteasome inhibitor. Inother cases, a combination therapy may include a CDK inhibitor incombination with a BCL-2 inhibitor.

In certain embodiments, a CDK inhibitor of the disclosure is representedby a compound disclosed in U.S. Pat. Nos. 7,271,193; 7,915,301;8,304,449; 7,884,127; 8,563,596, the entire contents of each of whichare incorporated herein by reference. In certain embodiments, a CDKinhibitor of the disclosure is represented by Formula I:

or a pharmaceutically acceptable salt thereof, wherein:

-   -   R₁ is optionally substituted phenyl;    -   R₂ and R₃ are each independently selected from hydroxy and OR₈,        wherein R₈ is optionally substituted C₁-C₁₀-alkyl;    -   R₄ is optionally substituted C₁-C₄-alkyl; and    -   R₉ is hydrogen or optionally substituted C₁-C₄-alkyl.

In certain embodiments, the compound or salt of Formula I is representedby Formula Ia:

In certain embodiments for a compound or salt of Formula I or Ia, R₁ isoptionally substituted with one or more substituents independentlyselected from hydroxy, cyano, halo, amino, C₁-C₄-alkyl, C₁-C₄-alkoxy,C₁-C₄-hydroxyalkyl, C₁-C₄-haloalkyl, and nitro. In certain embodiments,R₁ is substituted with one or more substituents independently selectedfrom hydroxy, cyano, halo, C₁-C₄-alkyl, and C₁-C₄-haloalkyl. In certainembodiments, R₁ is substituted with one or more substituentsindependently selected from halo and C₁-C₄-haloalkyl. In certainembodiments, R₁ is 2-chloro-4-trifluoromethylphenyl.

The term “alkyl” refers to a straight or branched hydrocarbon chainradical consisting solely of carbon and hydrogen atoms, and containingno unsaturation. In certain embodiments, an alkyl comprises one to eightcarbon atoms (i.e., C₁-C₈ alkyl). In other embodiments, an alkylcomprises one to five carbon atoms (i.e., C₁-C₅ alkyl). In otherembodiments, an alkyl comprises one to four carbon atoms (i.e., C₁-C₄alkyl). In other embodiments, an alkyl comprises one to three carbonatoms (i.e., C₁-C₃ alkyl). In other embodiments, an alkyl comprises oneto two carbon atoms (i.e., C₁-C₂ alkyl). In other embodiments, an alkylcomprises one carbon atom (i.e., C₁ alkyl). In other embodiments, analkyl comprises five to eight carbon atoms (i.e., C₅-C₈ alkyl). In otherembodiments, an alkyl comprises two to five carbon atoms (i.e., C₂-C₅alkyl). In other embodiments, an alkyl comprises three to five carbonatoms (i.e., C₃-C₅ alkyl). In certain embodiments, the alkyl group isselected from methyl, ethyl, 1-propyl (n-propyl), 1-methylethyl(iso-propyl), 1-butyl (n-butyl), 1-methylpropyl (sec-butyl),2-methylpropyl (iso-butyl), 1,1-dimethylethyl (tert-butyl), 1-pentyl(n-pentyl). The alkyl is attached to the rest of the molecule by asingle bond. Unless stated otherwise specifically in the specification,an alkyl group is optionally substituted by one or more substituentssuch as those substituents described herein.

The term “alkoxy” refers to a radical bonded through an oxygen atom ofthe formula —O— alkyl, where alkyl is an alkyl chain as defined above.

The term “amino” refers to the group —NR′R″, wherein R′ and R″ areindependently selected from hydrogen; and alkyl, hydroxyl, aryl,cycloalkyl, heterocycloalkyl, and heteroaryl, any one of which may beoptionally substituted with one or more substituents such as hydroxy,cyano, halo, amino, C₁-C₄-alkyl, C₁-C₄-alkoxy, C₁-C₄-hydroxyalkyl,C₁-C₄-haloalkyl, and nitro.

The term “C_(x-y)” when used in conjunction with a chemical moiety, suchas alkyl is meant to include groups that contain from x to y carbons inthe chain. For example, the term “C_(x-y)alkyl” refers to substituted orunsubstituted saturated hydrocarbon groups, including straight-chainalkyl and branched-chain alkyl groups that contain from x to y carbonsin the chain, including haloalkyl groups such as trifluoromethyl and2,2,2-trifluoroethyl, etc.

The term “haloalkyl” refers to an alkyl group that is substituted by oneor more halo radicals, for example, trifluoromethyl, difluoromethyl,fluoromethyl, 2,2,2-trifluoroethyl, 1-chloromethyl-2-fluoroethyl, andthe like. In some embodiments, the alkyl part of the haloalkyl isfurther optionally substituted as described herein.

The term “hydroxyalkyl” refers to an alkyl group that is substituted byone or more hydroxy radicals, for example, hydroxymethyl, hydroxyethyl,dihydroxymethyl, and the like. In some embodiments, the alkyl part ofthe hydroxyalkyl is further optionally substituted as described herein.

In certain embodiments for a compound or salt of Formula I or Ia, R₂ andR₃ are each independently selected from hydroxy and OR₈, wherein R₈ isC₁-C₁₀-alkyl optionally substituted with one or more substituentsindependently selected from hydroxy, cyano, halo, amino, ═O, ═S,C₁-C₄-alkoxy, and nitro. In certain embodiments, R₈ at each occurrenceis selected from optionally substituted C₁-C₆-alkyl, such as optionallysubstituted C₁-C₄-alkyl. In certain embodiments, R₂ and R₃ are eachindependently hydroxy.

In certain embodiments for a compound or salt of Formula I or Ia, R₄ isoptionally substituted C₁-C₄-alkyl, wherein R₄ is optionally substitutedwith one or more substituents selected from hydroxy, cyano, halo, amino,═O, ═S, C₁-C₄-alkoxy, and nitro. In certain embodiments, R₄ isoptionally substituted C₁-C₂-alkyl. In certain embodiments, R₄ ishydroxyalkyl, e.g., 2-hydroxymethyl.

In certain embodiments for a compound or salt of Formula I or Ia, R₉ isC₁-C₄-alkyl optionally substituted with hydroxy, cyano, halo, amino, ═O,═S, C₁-C₄-alkoxy, and nitro. In certain embodiments, R₉ is optionallysubstituted C₁-C₂-alkyl. In certain embodiments, R₉ is methyl. Incertain embodiments, R₉ is hydrogen.

In certain embodiments for a compound or salt of Formula I or Ia, acompound of Formula I is a compound or pharmaceutically acceptable saltselected from:(+)-trans-2-(2-Chloro-4-trifluoromethylphenyl)-8-(2-hydroxymethyl-1-methyl-pyrrolidin-3-yl)-5,7-dimethoxy-chromen-4-one;(+)-trans-2-(2-Chloro-4-trifluoromethylphenyl)-5,7-dihydroxy-8-(2-hydroxymethyl-1-methylpyrrolidin-3-yl)-chromen-4-one;and(+)-trans-2-(2-Chloro-4-trifluoromethylphenyl)-5,7-dihydroxy-8-(2-hydroxymethyl-1-methyl-pyrrolidin-3-yl)-chromen-4-onehydrochloride.

In certain embodiments, the compound of Formula I or Ia is representedby Formula Ib:

or a pharmaceutically acceptable salt thereof. In certain embodiments,the compound of Formula I, Ia, or Ib is in the form of an acid additionsalt, such as the hydrochloride salt.

The term “substituted” refers to moieties having substituents replacinga hydrogen on one or more carbons or heteroatoms of the structure. Itwill be understood that “substitution” or “substituted with” includesthe implicit proviso that such substitution is in accordance withpermitted valence of the substituted atom and the substituent, as wellas represents a stable compound, which does not readily undergotransformation such as rearrangement, cyclization, elimination, etc. Asused herein, the term “substituted” is contemplated to include allpermissible substituents of organic compounds. In a broad aspect, thepermissible substituents include acyclic and cyclic, branched andunbranched, carbocyclic and heterocyclic, aromatic and non-aromaticsubstituents of organic compounds. The permissible substituents can beone or more and the same or different for appropriate organic compounds.For purposes of this disclosure, the heteroatoms such as nitrogen mayhave hydrogen substituents and/or any permissible substituents oforganic compounds described herein which satisfy the valences of theheteroatoms.

Substituents can include any substituents described herein, for example,a halogen, a hydroxy, a carbonyl (such as a carboxyl, an alkoxycarbonyl,a formyl, or an acyl), a thiocarbonyl (such as a thioester, athioacetate, or a thioformate), an alkoxyl, a phosphoryl, a phosphate, aphosphonate, a phosphinate, an amino, an amido, an amidine, an imine, acyano, a nitro, an azido, a sulfhydryl, an alkylthio, a sulfate, asulfonate, a sulfamoyl, a sulfonamido, a sulfonyl, a heterocyclyl, anaralkyl, a carbocycle, a heterocycle, a cycloalkyl, a heterocycloalkyl,an aromatic and heteroaromatic moiety. In some embodiments, substituentsmay include any substituents described herein, for example: halogen,hydroxy, oxo (═O), thioxo (═S), cyano (—CN), nitro (—NO₂), imino (═N—H),oximo (═N—OH), hydrazino (═N—NH₂), —R^(b)—OR^(a), —R^(b)—OC(O)—R^(a),—R^(b)—OC(O)—OR^(a), —R^(b)—OC(O)—N(R^(a))₂, —R^(b)—N(R^(a))₂,—R^(b)—C(O)R^(a), —R^(b)—C(O)OR^(a), —R^(b)—C(O)N(R^(a))₂,—R^(b)—O—R^(c)—C(O)N(R^(a))₂, —R^(b)—N(R^(a))C(O)OR^(a),—R^(b)—N(R^(a))C(O)R^(a), —R^(b)—N(R^(a))S(O)_(t)R^(a) (where t is 1 or2), —R^(b)—S(O)_(t)R^(a) (where t is 1 or 2), —R^(b)—S(O)_(t)OR^(a)(where t is 1 or 2), and —R^(b)—S(O)_(t)N(R^(a))₂ (where t is 1 or 2);and alkyl, alkenyl, alkynyl, aryl, aralkyl, aralkenyl, aralkynyl,cycloalkyl, cycloalkylalkyl, heterocycloalkyl, heterocycloalkylalkyl,heteroaryl, and heteroarylalkyl any of which may be optionallysubstituted by alkyl, alkenyl, alkynyl, halogen, hydroxy, haloalkyl,haloalkenyl, haloalkynyl, oxo (═O), thioxo (═S), cyano (—CN), nitro(—NO₂), imino (═N—H), oximo (═N—OH), hydrazine (═N—NH₂), —R^(b)—OR^(a),—R^(b)—OC(O)—R^(a), —R^(b)—OC(O)—OR^(a), —R^(b)—OC(O)—N(R^(a))₂,—R^(b)—N(R^(a))₂, —R^(b)—C(O)R^(a), —R^(b)—C(O)OR^(a),—R^(b)—C(O)N(R^(a))₂, —R^(b)—O—R^(c)—C(O)N(R^(a))₂,—R^(b)—N(R^(a))C(O)OR^(a), —R^(b)—N(R^(a))C(O)R^(a),—R^(b)—N(R^(a))S(O)_(t)R^(a) (where t is 1 or 2), —R^(b)—S(O)_(t)R^(a)(where t is 1 or 2), —R^(b)—S(O)_(t)OR^(a) (where t is 1 or 2) and—R^(b)—S(O)_(t)N(R^(a))₂ (where t is 1 or 2); wherein each R^(a) isindependently selected from hydrogen, alkyl, cycloalkyl,cycloalkylalkyl, aryl, aralkyl, heterocycloalkyl, heterocycloalkylalkyl,heteroaryl, or heteroarylalkyl, wherein each R^(a), valence permitting,may be optionally substituted with alkyl, alkenyl, alkynyl, halogen,haloalkyl, haloalkenyl, haloalkynyl, oxo (═O), thioxo (═S), cyano (—CN),nitro (—NO₂), imino (═N—H), oximo (═N—OH), hydrazine (═N—NH₂),—R^(b)—OR^(a), —R^(b)—OC(O)—R^(a), —R^(b)—OC(O)—OR^(a),—R^(b)—OC(O)—N(R^(a))₂, —R^(b)—N(R^(a))₂, —R^(b)—C(O)R^(a),—R^(b)—C(O)OR^(a), —R^(b)—C(O)N(R^(a))₂, —R^(b)—O—R^(c)—C(O)N(R^(a))₂,—R^(b)—N(R^(a))C(O)OR^(a), —R^(b)—N(R^(a))C(O)R^(a),—R^(b)—N(R^(a))S(O)_(t)R^(a) (where t is 1 or 2), —R^(b)—S(O)_(t)R^(a)(where t is 1 or 2), —R^(b)—S(O)_(t)OR^(a) (where t is 1 or 2) and—R^(b)—S(O)_(t)N(R^(a))₂ (where t is 1 or 2); and wherein each R^(b) isindependently selected from a direct bond or a straight or branchedalkylene, alkenylene, or alkynylene chain, and each R^(c) is a straightor branched alkylene, alkenylene or alkynylene chain.

Procedures for the manufacture of the compounds of Formula I, Ia, and Ibor the pharmaceutically acceptable salts thereof, may be found in PCTPatent Publication No. WO2004004632 (corresponding to U.S. Pat. No.7,271,193) and PCT Patent Publication No. WO2007148158.

The present disclosure provides pharmaceutically-acceptable salts of anycompound described herein, e.g., a compound of Formula I, Ia, Ib, BCL-2inhibitors and proteasome inhibitors. Pharmaceutically-acceptable saltsinclude, for example, acid-addition salts and base-addition salts. Theacid that is added to a compound to form an acid-addition salt can be anorganic acid or an inorganic acid. A base that is added to a compound toform a base-addition salt can be an organic base or an inorganic base.In some cases, a pharmaceutically-acceptable salt is a metal salt. Insome cases, a pharmaceutically-acceptable salt is an ammonium salt.

Acid addition salts can arise from the addition of an acid to a compounddescribed herein. In some cases, the acid is organic. In some cases, theacid is inorganic. Non-limiting examples of suitable acids includehydrochloric acid, hydrobromic acid, hydroiodic acid, nitric acid,nitrous acid, sulfuric acid, sulfurous acid, a phosphoric acid,nicotinic acid, isonicotinic acid, lactic acid, salicylic acid,4-aminosalicylic acid, tartaric acid, ascorbic acid, gentisinic acid,gluconic acid, glucaronic acid, saccaric acid, formic acid, benzoicacid, glutamic acid, pantothenic acid, acetic acid, propionic acid,butyric acid, fumaric acid, succinic acid, citric acid, oxalic acid,maleic acid, hydroxymaleic acid, methylmaleic acid, glycolic acid, malicacid, cinnamic acid, mandelic acid, 2-phenoxybenzoic acid,2-acetoxybenzoic acid, embonic acid, phenylacetic acid,N-cyclohexylsulfamic acid, methanesulfonic acid, ethanesulfonic acid,benzenesulfonic acid, p-toluenesulfonic acid, 2-hydroxyethanesulfonicacid, ethane-1,2-disulfonic acid, 4-methylbenzenesulfonic acid,naphthalene-2-sulfonic acid, naphthalene-1,5-disulfonic acid,2-phosphoglyceric acid, 3-phosphoglyceric acid, glucose-6-phosphoricacid, and an amino acid.

Metal salts can arise from the addition of an inorganic base to acompound of the invention. The inorganic base consists of a metal cationpaired with a basic counterion, such as, for example, hydroxide,carbonate, bicarbonate, or phosphate. The metal can be an alkali metal,alkaline earth metal, transition metal, or main group metal. In someembodiments, the metal is lithium, sodium, potassium, cesium, cerium,magnesium, manganese, iron, calcium, strontium, cobalt, titanium,aluminum, copper, cadmium, or zinc.

In some embodiments, a metal salt is a lithium salt, a sodium salt, apotassium salt, a cesium salt, a cerium salt, a magnesium salt, amanganese salt, an iron salt, a calcium salt, a strontium salt, a cobaltsalt, a titanium salt, an aluminum salt, a copper salt, a cadmium salt,or a zinc salt.

Ammonium salts can arise from the addition of ammonia or an organicamine to a compound described herein. Non-limiting examples of suitableorganic amines include triethyl amine, diisopropyl amine, ethanol amine,diethanol amine, triethanol amine, morpholine, N-methylmorpholine,piperidine, N-methylpiperidine, N-ethylpiperidine, dibenzyl amine,piperazine, pyridine, pyrrazole, pipyrrazole, imidazole, pyrazine,pipyrazine, ethylenediamine, N,N′-dibenzylethylene diamine, procaine,chloroprocaine, choline, dicyclohexyl amine, and N-methylglucamine.

Non-limiting examples of suitable ammonium salts include is a triethylamine salt, a diisopropyl amine salt, an ethanol amine salt, a diethanolamine salt, a triethanol amine salt, a morpholine salt, anN-methylmorpholine salt, a piperidine salt, an N-methylpiperidine salt,an N-ethylpiperidine salt, a dibenzyl amine salt, a piperazine salt, apyridine salt, a pyrrazole salt, a pipyrrazole salt, an imidazole salt,a pyrazine salt, a pipyrazine salt, an ethylene diamine salt, anN,N′-dibenzylethylene diamine salt, a procaine salt, a chloroprocainesalt, a choline salt, a dicyclohexyl amine salt, and a N-methylglucaminesalt.

Non-limiting examples of suitable acid addition salts include ahydrochloride salt, a hydrobromide salt, a hydroiodide salt, a nitratesalt, a nitrite salt, a sulfate salt, a sulfite salt, a phosphate salt,a hydrogen phosphate salt, a dihydrogen phosphate salt, a carbonatesalt, a bicarbonate salt, a nicotinate salt, an isonicotinate salt, alactate salt, a salicylate salt, a 4-aminosalicylate salt, a tartratesalt, an ascorbate salt, a gentisinate salt, a gluconate salt, aglucaronate salt, a saccarate salt, a formate salt, a benzoate salt, aglutamate salt, a pantothenate salt, an acetate salt, a propionate salt,a butyrate salt, a fumarate salt, a succinate salt, a citrate salt, anoxalate salt, a maleate salt, a hydroxymaleate salt, a methylmaleatesalt, a glycolate salt, a malate salt, a cinnamate salt, a mandelatesalt, a 2-phenoxybenzoate salt, a 2-acetoxybenzoate salt, an embonatesalt, a phenylacetate salt, an N-cyclohexylsulfamate salt, amethanesulfonate salt, an ethanesulfonate salt, a benzenesulfonate salt,a p-toluenesulfonate salt, a 2-hydroxyethanesulfonate salt, anethane-1,2-disulfonate salt, a 4-methylbenzenesulfonate salt, anaphthalene-2-sulfonate salt, a naphthalene-1,5-disulfonate salt, a2-phosphoglycerate salt, a 3-phosphoglycerate salt, aglucose-6-phosphate salt, and an amino acid salt.

The compounds described herein, e.g., the compounds and salts ofFormulas I, Ia, Ib, BCL-2 inhibitors and proteasome inhibitors, may insome cases exist as diastereomers, enantiomers, or other stereoisomericforms. The compounds presented herein include all diastereomeric,enantiomeric, and epimeric forms as well as the appropriate mixturesthereof. Separation of stereoisomers may be performed by chromatographyor by forming diastereomers and separating by recrystallization, orchromatography, or any combination thereof. (Jean Jacques, Andre Collet,Samuel H. Wilen, “Enantiomers, Racemates and Resolutions”, John WileyAnd Sons, Inc., 1981, herein incorporated by reference for thisdisclosure). Stereoisomers may also be obtained by stereoselectivesynthesis.

The compounds described herein, e.g., the compounds and salts ofFormulas I, Ia, Ib, BCL-2 inhibitors and proteasome inhibitors, includethe use of amorphous forms as well as crystalline forms (also known aspolymorphs). The compounds described herein may be in the form ofpharmaceutically acceptable salts. As well, active metabolites of thesecompounds having the same type of activity are included in the scope ofthe present disclosure. In addition, the compounds described herein canexist in unsolvated as well as solvated forms with pharmaceuticallyacceptable solvents such as water, ethanol, and the like. The solvatedforms of the compounds presented herein are also considered to bedisclosed herein.

The compounds described herein, e.g., the compounds and salts ofFormulas I, Ia, Ib, BCL-2 inhibitors and proteasome inhibitors, includecompounds that exhibit their natural isotopic abundance, and compoundswhere one or more of the atoms are artificially enriched in a particularisotope having the same atomic number, but an atomic mass or mass numberdifferent from the atomic mass or mass number predominantly found innature. All isotopic variations of the compounds of the presentinvention, whether radioactive or not, are encompassed within the scopeof the present invention. For example, hydrogen has three naturallyoccurring isotopes, denoted ¹H (protium), ²H (deuterium), and ³H(tritium). Protium is the most abundant isotope of hydrogen in nature.Enriching for deuterium may afford certain therapeutic advantages, suchas increased in vivo half-life and/or exposure, or may provide acompound useful for investigating in vivo routes of drug elimination andmetabolism. Isotopically-enriched compounds may be prepared.

Compounds described herein, e.g., the compounds and salts of Formulas I,Ia, Ib, BCL-2 inhibitors and proteasome inhibitors, wherein the compoundhas carbon-carbon double bonds or carbon-nitrogen double bonds mayexist, where applicable, in Z- or E-form (or cis- or trans-form).Furthermore, some chemical entities may exist in various tautomericforms. Unless otherwise specified, chemical entities described hereinare intended to include all Z-, E- and tautomeric forms as well.

In certain cases, a compound described herein may be a prodrug, e.g.,wherein a carboxylic acid present in the parent compound is presented asan ester. The term “prodrug” is intended to encompass compounds which,under physiologic conditions, are converted into pharmaceutical agents,i.e., parent compound, of the present disclosure. One method for makinga prodrug is to include one or more selected moieties which arehydrolyzed under physiologic conditions to reveal the desired molecule.In certain embodiments, the prodrug is converted by an enzymaticactivity of the host animal such as enzymatic activity in specifictarget cells in the host animal. For example, esters or carbonates(e.g., esters or carbonates of alcohols or carboxylic acids) arepreferred prodrugs of the present disclosure.

Prodrugs are often useful because, in some situations, they may beeasier to administer than the parent drug. They may, for instance, bebioavailable by oral administration whereas the parent is not. Prodrugsmay help enhance the cell permeability of a compound relative to theparent drug. For example, the prodrug may have improved cellpermeability over the parent compound. The prodrug may also haveimproved solubility in pharmaceutical formulations over the parent drug.In some embodiments, the design of a prodrug increases the lipophilicityof the pharmaceutical agent. In some embodiments, the design of aprodrug increases the effective water solubility.

In certain embodiments, a cyclin-dependent kinase (CDK) inhibitor, e.g.,a compound or salt of Formula I, Ia or Ib, may be used in combinationwith an inhibitor of one or more proteins in the BCL-2 family.Inhibitors of BCL-2 anti-apoptotic family of proteins alter at least acell survival pathway. Apoptosis activation may occur via an extrinsicpathway triggered by the activation of cell surface death receptors oran intrinsic pathway triggered by developmental cues and diverseintracellular stresses. This intrinsic pathway, also known as the stresspathway or mitochondrial pathway, is primarily regulated by the BCL-2family, a class of key regulators of caspase activation consisting ofanti-apoptotic (pro-survival) proteins having BH1-BH4 domains (BCL-2,i.e., the BCL-2 protein member of the BCL-2 anti-apoptotic proteinfamily), BCL-xL, BCL-w, A1, MCL-1, and BCL-B); pro-apoptotic proteinshaving BH1, BH2, and BH3 domains (BAX, BAK, and BOK); and pro-apoptoticBH3-only proteins (BIK, BAD, BID, BIM, BMF, HRK, NOXA, and PUMA) (see,e.g., Cory et al., Nature Reviews Cancer 2:647-56 (2002); Cory et al.,Cancer Cell 8:5-6 (2005); Adams et al., Oncogene 26:1324-1337 (2007)).BCL-2 anti-apoptotic proteins block activation of pro-apoptoticmulti-domain proteins BAX and BAK (see, e.g., Adams et al., Oncogene26:1324-37 (2007)).

As used herein, the term “BCL-2 inhibitor” refers to an agent that iscapable of inhibiting one or more proteins in the BCL-2 family ofanti-apoptotic proteins, e.g., BCL-2, BCL-xL, and BCL-w. In certainembodiments, a BCL-2 inhibitor of the disclosure inhibits one protein ofthe BCL-2 family selectively, e.g., a BCL-2 inhibitor may selectivelyinhibit BCL-2 and not BCL-xl or BCL-w.

The BCL-2 inhibitor described herein may inhibit one or more of BCL-2,BCL-xL, and BCL-w. In certain embodiments, the inhibitor of BCL-2anti-apoptotic family of proteins inhibits BCL-2. In certainembodiments, the inhibitor of BCL-2 anti-apoptotic family of proteinsinhibits BCL-2 and does not inhibit other members of the BCL-2 family ofproteins, e.g., does not inhibit BCL-xL or BCL-w. In certainembodiments, the BCL-2 inhibitor is a BH3-mimetic.

In certain embodiments, the BCL-2 inhibitor of the disclosure inhibitsBCL-xL function. In addition to inhibition of BCL-xL, the inhibitor mayalso interact with and/or inhibit one or more functions of BCL-2, e.g.,BCL-xL/BCL-2 inhibitors. In certain embodiments, a BCL-2 inhibitor ofthe disclosure inhibits each of BCL-xL and BCL-w. In certainembodiments, a BCL-2 inhibitor of the disclosure inhibits BCL-xL, BCL-2,and BCL-w.

In certain embodiments, a BCL-2 inhibitor interferes with theinteraction between the BCL-2 anti-apoptotic protein family member andone or more ligands or receptors to which the BCL-2 anti-apoptoticprotein family member would bind in the absence of the inhibitor. Inother embodiments, an inhibitor of one or more BCL-2 anti-apoptoticprotein family members, wherein the inhibitor inhibits at least oneBCL-2 protein specifically, binds only to one or more of BCL-xL, BCL-2,BCL-w and not to other Bcl-2 anti-apoptotic Bcl-2 family members, suchas Mcl-1 and BCL2A1.

Binding affinity of a BCL-2 inhibitor for BCL-2 family proteins may bemeasured. By way of example, binding affinity of a BCL-xL inhibitor maybe determined using a competition fluorescence polarization assay inwhich a fluorescent BAK BH3 domain peptide is incubated with BCL-xLprotein (or other BCL-2 family protein) in the presence or absence ofincreasing concentrations of the BCL-XL inhibitor as previouslydescribed (see, e.g., U.S. Patent Publication 20140005190; Park et al.,Cancer Res. 73:5485-96 (2013); Wang et al., Proc. Natl. Acad. Sci USA97:7124-9 (2000); Zhang et al., Anal Biochem, 307:70-5 (2002); Brunckoet al., J. Med. Chem, 50:641-62 (2007)). Percent inhibition may bedetermined by the equation: 1-[(mP value of well—negativecontrol)/range)]×100%. Inhibitory constant (K_(i)) value is determinedby the formula: K_(i)=[I]₅₀/([L]₅₀/K_(d)+[P]₀/K_(d)+1) as described inBruncko et al., J. Med. Chem. 50:641-62 (2007) (see, also, Wang, FEBSLett. 360:111-114 (1995)).

Examples of BCL-2 inhibitors include ABT-263(4-[4-[[2-(4-chlorophenyl)-5,5-dimethylcyclohexen-1-yl]methyl]piperazin-1-yl]-N-[4-[[(2R)-4-morpholin-4-yl-1-phenylsulfanylbutan-2-yl]amino]-3-(trifluoromethylsulfonyl)phenyl]sulfonylbenzamideor IUPAC,(R)-4-(4-((4′-chloro-4,4-dimethyl-3,4,5,6-tetrahydro-[1,1′-biphenyl]-2-yl)methyl)piperazin-1-yl)-N-((4-((4-morpholino-1-(phenylthio)butan-2-yl)amino)-3-((trifluoromethyl)sulfonyl)phenyl)sulfonyl)benzamide)(see, e.g., Park et al., 2008, J. Med. Chem. 51:6902; Tse et al., CancerRes., 2008, 68:3421; Int'l Patent Appl. Pub. No. WO 2009/155386; U.S.Pat. Nos. 7,390,799, 7,709,467, 7,906,505, 8,624,027) and ABT-737(4-[4-[(4′-Chloro[1,1′-biphenyl]-2-yl)methyl]-1-piperazinyl]-N-[[4-[[(1R)-3-(dimethylamino)-1-[(phenylthio)methyl]propyl]amino]-3-nitrophenyl]sulfonyl]benzamide,Benzamide,4-[4-[(4′-chloro[1,1′-biphenyl]-2-yl)methyl]-1-piperazinyl]-N-[[4-[[(1R)-3-(dimethylamino)-1-[(phenylthio)methyl]propyl]amino]-3-nitrophenyl]sulfonyl]-or4-[4-[[2-(4-chlorophenyl)phenyl]methyl]piperazin-1-yl]-N-[4-[[(2R)-4-(dimethylamino)-1-phenylsulfanylbutan-2-yl]amino]-3-nitrophenyl]sulfonylbenzamide)(see, e.g., Oltersdorf et al., Nature, 2005, 435:677; U.S. Pat. No.7,973,161; U.S. Pat. No. 7,642,260).

In other embodiments, the BCL-2 inhibitor is a quinazoline sulfonamidecompound (see, e.g., Sleebs et al., 2011, J. Med. Chem. 54:1914). Instill another embodiment, the BCL-inhibitor is a small molecule compoundas described in Zhou et al., J. Med. Chem., 2012, 55:4664 (see, e.g.,Compound 21(R)-4-(4-chlorophenyl)-3-(3-(4-(4-(4-((4-(dimethylamino)-1-(phenylthio)butan-2-yl)amino)-3-nitrophenylsulfonamido)phenyl)piperazin-1-yl)phenyl)-5-ethyl-1-methyl-1H-pyrrole-2-carboxylicacid) and Zhou et al., J. Med. Chem., 2012, 55:6149 (see, e.g., Compound14(R)-5-(4-Chlorophenyl)-4-(3-(4-(4-(4-((4-(dimethylamino)-1-(phenylthio)butan-2-yl)amino)-3-nitrophenylsulfonamido)phenyl)piperazin-1-yl)phenyl)-1-ethyl-2-methyl-1H-pyrrole-3-carboxylicacid; Compound 15(R)-5-(4-Chlorophenyl)-4-(3-(4-(4-(4-((4-(dimethylamino)-1-(phenylthio)butan-2-yl)amino)-3-nitrophenylsulfonamido)phenyl)piperazin-1-yl)phenyl)-1-isopropyl-2-methyl-1H-pyrrole-3-carboxylicacid). In other embodiments, the BCL-inhibitor is a BCL-2/BCL-xLinhibitor such as BM-1074 (see, e.g., Aguilar et al., 2013, J. Med.Chem. 56:3048); BM-957 (see, e.g., Chen et al., 2012, J. Med. Chem.55:8502); BM-1197 (see, e.g., Bai et al., PLoS One 2014 Jun.5;9(6):e99404. Doi: 10.1371/journal.pone. 009904); U.S. Patent Appl. No.2014/0199234; N-acylsufonamide compounds (see, e.g., Int'l Patent Appl.Pub. No. WO 2002/024636, Int'l Patent Appl. Pub. No. WO 2005/049593,Int'l Patent Appl. Pub. No. WO 2005/049594, U.S. Pat. No. 7,767,684,U.S. Pat. No. 7,906,505). In still another embodiment, the BCL-2inhibitor is a small molecule macrocyclic compound (see, e.g., Int'lPatent Appl. Pub. No. WO 2006/127364, U.S. Pat. No. 7,777,076). In yetanother embodiment, the BCL-2 inhibitor is an isoxazolidine compound(see, e.g., Int'l Patent Appl. Pub. No. WO 2008/060569, U.S. Pat. No.7,851,637, U.S. Pat. No. 7,842,815). In yet another embodiment, theBCL-2 inhibitor is S44563 (see, e.g., Loriot et. al., Cell Death andDisease, 2014, 5, e1423). In one embodiment, the BCL-2 inhibitor is(R)-3-((4′-chloro-[1,1′-biphenyl]-2-yl)methyl)-N-((4-(((R)-4-(dimethylamino)-1-(phenylthio)butan-2-yl)amino)-3-nitrophenyl)sulfonyl)-2,3,4,4a,5,6-hexahydro-1H-pyrazino[1,2-a]quinoline-8-carboxamide.In another embodiment, the BCL-2 inhibitor is a small moleculeheterocyclic compounds (see, e.g., U.S. Pat. No. 9,018,381).

In certain cases, a BCL-2 inhibitor is used in combination with acompound or salt of Formula I, Ia or Ib. Any BCL-2 inhibitor may be usedand may exhibit a synergistic effect when used in combination with acompound or salt of Formula I, Ia or Ib. A BCL-2 family inhibitor mayinhibit one or more members of the BCL-2 family, including Bcl-2,Bcl-xL, Bcl-w, BAK1, BAX, BCL2, BCL2A1, BCL2L1, BCL2L2, BCL2L10,BCL2L13, BCL2L14, BOK and MCL1. In certain embodiments, a compound orsalt of Formula I, Ia or Ib is used in combination with any of thefollowing: navitoclax, venetoclax, A-1155463, A-1331852, ABT-737,obatoclax, TW-37, A-1210477, AT101, HA14-1, BAM7, sabutoclax, UMI-77,gambogic acid, maritoclax, MIM1, methylprednisolone, iMAC2, Baxinhibitor peptide V5, Bax inhibitor peptide P5, Bax channel blocker, andARRY 520 trifluoroacetate. In some examples, voruciclib is used incombination with navitoclax. In certain embodiments, voruciclib is usedin combination with venetoclax.

In some embodiments, a BCL-2 inhibitor is used in combination with a CDKinhibitor of the disclosure, e.g., a compound of Formula I, Ia or Ib,for the treatment of a blood cancer. In certain embodiments, the bloodcancer is leukemia, such as acute myeloid leukemia (AML), chronicmyeloid leukemia (CML), acute lymphocytic lymphoma (ALL), and chroniclymphocytic leukemia (CLL). In certain embodiments, the blood cancer isa non-Hodgkin lymphoma, such as B-cell or T-cell lymphoma. B-celllymphomas include diffuse large B-cell lymphoma (DLBCL), primarymediastinal B-cell lymphoma, intravascular large B-cell lymphoma,follicular lymphoma, small lymphocytic lymphomia (SLL), mantle celllymphoma, marginal zone B-cell lymphomas, extranodal marginal zoneB-cell lymphomas, nodal marginal zone B-cell lymphoma, splenic marginalzone B-cell lymphoma, Burkitt lymphoma, lymphoplasmacytic lymphoma, andprimary central nervous system lymphoma. T-cell lymphomas includeprecursor T-lymphoblastic lymphoma, peripheral T-cell lymphomas,cutaneous T-cell lymphomas, adult T-cell lymphoma with subtypes:smoldering chronic, acute, and lymphoma, angioimmunoblastic T-celllymphoma, extranodal natural killer/T-cell lymphoma, nasal type,enteropathy-associated intestinal T-cell lymphoma (EATL) with subtypes Iand II, and anaplastic large cell lymphoma (ALCL). Combinations of thepresent disclosure, e.g., combinations of CDK inhibitors and BCL-2inhibitors described herein, may be used to treat a blood cancerdescribed herein.

The terms “treat,” “treating” or “treatment,” as used herein, mayinclude alleviating, abating or ameliorating a disease or conditionsymptoms, preventing additional symptoms, ameliorating or preventing theunderlying causes of symptoms, inhibiting the disease or condition,e.g., arresting the development of the disease or condition, relievingthe disease or condition, causing regression of the disease orcondition, relieving a condition caused by the disease or condition, orstopping the symptoms of the disease or condition eitherprophylactically and/or therapeutically.

The disclosure provides methods of preventing, or reducing, a relapse ofa cancer in a subject in need thereof. In certain embodiments, the term“prevent” or “preventing” as related to a disease or disorder may referto a compound or combination that, in a statistical sample, reduces theoccurrence of the disorder or condition in the treated sample relativeto an untreated control sample, or delays the onset or reduces theseverity of one or more symptoms of the disorder or condition relativeto the untreated control sample. The method includes administering acombination therapy described herein to treat minimal residual disease,and/or as maintenance therapy, e.g., as a prolonged or extended therapyafter cessation of another cancer treatment. For example, thecombination therapy may be administered after cessation of anothercancer therapy, such as chemotherapy, radiation therapy and/or surgery.

In certain aspects, a proteasome inhibitor may be combined or used incombination with a CDK inhibitor of the disclosure, e.g., a compound orsalt of any one of Formulas I, Ia, or Ib. In eukaryotic cells, theubiquitin (Ub)—proteasome pathway (UPS) involves Ub modification andsubsequent degradation of protein substrates. UPS controls the levels ofmany cellular regulatory proteins, including transcription factors, cellcycle regulatory proteins and factors participating in a variety ofcellular processes. The common feature of UPS pathway is that the highlyconserved Ub is covalently attached to the target proteins through aseries of enzymes, namely E1 Ub-activating enzyme, E2 Ub-conjugatingenzyme and E3 Ub ligase. The E1 first activates Ub and transfers it toE2. From the E2 enzyme, the Ub is transferred directly to the targetprotein or indirectly through an E3 Ub ligase. The polyubiquitylatedprotein is recognized and degraded by 26S proteasome, a large complexwith multiple proteolytic activities.

As used herein, the term “proteasome inhibitor” refers to an agent thatblocks the action of a proteasome. Proteasome inhibition may preventdegradation of pro-apoptotic factors such as the p53 protein, permittingactivation of programmed cell death in neoplastic cells dependent uponsuppression of pro-apoptotic pathways.

Any proteasome inhibitor may be used and may exhibit a synergisticeffect when used in combination with a CDK inhibitor, e.g., a compoundor salt of Formula I, Ia, or Ib. Non-limiting examples of proteasomeinhibitors may include: bortezomib, marizomib, ixazomib, disulfiram,epigallocatechin-3-gallate, salinosporamide A, carfilzomib, ONX 0912,CEP-18770, MLN9708, epoxomicin, and MG132.

In some embodiments, a proteasome inhibitor is used in combination witha CDK inhibitor of the disclosure, e.g., a compound of Formula I, Ia orIb, for the treatment of a blood cancer, such as diffuse large B-celllymphoma or triple negative breast cancer.

In some aspects, combinations described herein, e.g., combinations ofCDK inhibitors with BCL-2 inhibitors or proteasome inhibitors, can beutilized for the treatment of cancer. A combination therapy describedherein can reduce the likelihood of metastasis in a subject in needthereof. In some embodiments, the metastasis is a solid tumor. In someembodiments, the metastasis is a liquid tumor. Cancers that are liquidtumors can be those that occur, for example, in blood, bone marrow, andlymph nodes, and can include, for example, leukemia, myeloid leukemia,lymphocytic leukemia, lymphoma, Hodgkin's lymphoma, melanoma, andmultiple myeloma. Leukemias include, for example, acute lymphoblasticleukemia (ALL), acute myeloid leukemia (AML), chronic lymphocyticleukemia (CLL), chronic myelogenous leukemia (CML), and hairy cellleukemia. Cancers that are solid tumors include, for example, prostatecancer, testicular cancer, breast cancer, brain cancer, pancreaticcancer, colon cancer, thyroid cancer, stomach cancer, lung cancer,ovarian cancer, Kaposi's sarcoma, skin cancer, squamous cell skincancer, renal cancer, head and neck cancers, throat cancer, squamouscarcinomas that form on the moist mucosal linings of the nose, mouth,throat, bladder cancer, osteosarcoma, cervical cancer, endometrialcancer, esophageal cancer, liver cancer, and kidney cancer. In someembodiments, the condition treated by the methods described herein ismetastasis of melanoma cells, prostate cancer cells, testicular cancercells, breast cancer cells, brain cancer cells, pancreatic cancer cells,colon cancer cells, thyroid cancer cells, stomach cancer cells, lungcancer cells, ovarian cancer cells, Kaposi's sarcoma cells, skin cancercells, renal cancer cells, head or neck cancer cells, throat cancercells, squamous carcinoma cells, bladder cancer cells, osteosarcomacells, cervical cancer cells, endometrial cancer cells, esophagealcancer cells, liver cancer cells, or kidney cancer cells.

The methods described herein can also be used for inhibiting progressionof metastatic cancer tumors. Non-limiting examples of cancers includeadrenocortical carcinoma, childhood adrenocortical carcinoma,AIDS-related cancers, anal cancer, appendix cancer, basal cellcarcinoma, childhood basal cell carcinoma, bladder cancer, childhoodbladder cancer, bone cancer, brain tumor, childhood astrocytomas,childhood brain stem glioma, childhood central nervous system atypicalteratoid/rhabdoid tumor, childhood central nervous system embryonaltumors, childhood central nervous system germ cell tumors, childhoodcraniopharyngioma brain tumor, childhood ependymoma brain tumor, breastcancer, childhood bronchial tumors, carcinoid tumor, childhood carcinoidtumor, gastrointestinal carcinoid tumor, carcinoma of unknown primary,childhood carcinoma of unknown primary, childhood cardiac tumors,cervical cancer, childhood cervical cancer, childhood chordoma , chronicmyeloproliferative disorders, colon cancer, colorectal cancer, childhoodcolorectal cancer, extrahepatic bile duct cancer, ductal carcinoma insitu (DCIS), endometrial cancer, esophageal cancer, childhood esophagealcancer, childhood esthesioneuroblastoma, eye cancer, malignant fibroushistiocytoma of bone, gallbladder cancer, gastric (stomach) cancer,childhood gastric cancer, gastrointestinal stromal tumors (GIST),childhood gastrointestinal stromal tumors (GIST), childhood extracranialgerm cell tumor, extragonadal germ cell tumor, gestational trophoblastictumor, glioma, head and neck cancer, childhood head and neck cancer,hepatocellular cancer, hypopharyngeal cancer, kidney cancer, renal cellkidney cancer, Wilms tumor, childhood kidney tumors, Langerhans cellhistiocytosis, laryngeal cancer, childhood laryngeal cancer, leukemia,acute lymphoblastic leukemia (ALL), acute myeloid leukemia (AML),chronic lymphocytic leukemia (CLL), chronic myelogenous leukemia (cml),hairy cell leukemia, lip cancer, liver cancer (primary), childhood livercancer (primary), lobular carcinoma in situ (LCIS), lung cancer,non-small cell lung cancer, small cell lung cancer, lymphoma,AIDS-related lymphoma, burkitt lymphoma, cutaneous t-cell lymphoma,Hodgkin lymphoma, non-Hodgkin lymphoma, primary central nervous systemlymphoma (CNS), melanoma, childhood melanoma, intraocular melanoma,Merkel cell carcinoma, malignant mesothelioma, childhood malignantmesothelioma, metastatic squamous neck cancer with occult primary,midline tract carcinoma involving NUT gene, mouth cancer, childhoodmultiple endocrine neoplasia syndromes, mycosis fungoides,myelodysplastic syndromes, myelodysplastic neoplasms, myeloproliferativeneoplasms, multiple myeloma, nasal cavity cancer, nasopharyngeal cancer,childhood nasopharyngeal cancer, neuroblastoma, oral cancer, childhoodoral cancer, oropharyngeal cancer, ovarian cancer, childhood ovariancancer, epithelial ovarian cancer, low malignant potential tumor ovariancancer, pancreatic cancer, childhood pancreatic cancer, pancreaticneuroendocrine tumors (islet cell tumors), childhood papillomatosis,paraganglioma, paranasal sinus cancer, parathyroid cancer, penilecancer, pharyngeal cancer, pheochromocytoma, pituitary tumor, plasmacell neoplasm , childhood pleuropulmonary blastoma, prostate cancer,rectal cancer, renal pelvis transitional cell cancer, retinoblastoma,salivary gland cancer, childhood salivary gland cancer, Ewing sarcomafamily of tumors, Kaposi Sarcoma, osteosarcoma, rhabdomyosarcoma,childhood rhabdomyosarcoma, soft tissue sarcoma, uterine sarcoma, Sézarysyndrome, childhood skin cancer, nonmelanoma skin cancer, smallintestine cancer, squamous cell carcinoma, childhood squamous cellcarcinoma, testicular cancer, childhood testicular cancer, throatcancer, thymoma and thymic carcinoma, childhood thymoma and thymiccarcinoma, thyroid cancer, childhood thyroid cancer, ureter transitionalcell cancer, urethral cancer, endometrial uterine cancer, vaginalcancer, vulvar cancer, and Waldenström macroglobulinemia.

The combination therapies described herein may be used together withother therapies such as radiation therapy. Chemotherapy and radiotherapytreatment regimens can comprise a finite number of cycles of on-drugtherapy followed by off-drug therapy, or comprise a finite timeframe inwhich the chemotherapy or radiotherapy is administered. The protocolscan be determined by clinical trials, drug labels, and clinical staff inconjunction with the subject to be treated. The number of cycles of achemotherapy or radiotherapy or the total length of time of achemotherapy or radiotherapy regimen can vary depending on the subject'sresponse to the cancer therapy. A pharmaceutical agent described hereincan be administered after the treatment regimen of chemotherapy orradiotherapy has been completed.

In some aspects, the combinations described herein can be utilized totreat a subject in need thereof. In some cases, the subject to betreated by methods and compositions disclosed herein can be a humansubject. A subject to be treated by methods and compositions disclosedherein can be a non-human animal. Non-limiting examples of non-humananimals can include a non-human primate, a livestock animal, a domesticpet, and a laboratory animal.

In certain embodiments, the combination therapies described herein maybe administered as separate agents or may be combined into a singlepharmaceutical composition. For example, a combination of a CDKinhibitor, e.g., a compound or salt of Formula I, Ia, or Ib, and a BCL-2inhibitor, e.g., venetoclax or navitoclax, may be formulated as twoseparate pharmaceutical compositions or the two agents may beco-formulated as a single pharmaceutical composition.

In certain embodiments, a CDK inhibitor, e.g., a compound or salt ofFormula I, Ia, or Ib, is co-formulated with a BCL-2 inhibitor orproteasome inhibitor. In some cases, a compound of Formula I, Ia, or Ibis co-formulated with any one of navitoclax, venetoclax, bortezomib,marizomib or ixazomib or a combination thereof.

In certain embodiments, the disclosure provides a pharmaceuticalcomposition, e.g., for oral or parenteral administration, comprising acompound or salt of Formula I, Ia, or Ib. In some aspects, thepharmaceutical composition comprises a compound or salt of Formula I,Ia, or Ib in an amount of at least about 1 mg to about 1000 mg, fromabout 100 mg to about 400 mg, from about 100 mg to about 200 mg, fromabout 200 mg to about 400 mg, or from about 250 mg to about 350 mg. Forexample, a pharmaceutical composition of the disclosure may compriseabout 100 mg, about 120 mg, about 140 mg, about 160 mg, about 180 mg,about 200 mg, about 220 mg, about 240 mg, about 260 mg, about 280 mg,about 300 mg, about 320 mg, about 340 mg, about 360 mg, about 380 mg,about 400 mg, about 420 mg, about 440 mg, about 460 mg, about 480 mg, orabout 500 mg of a compound of Formula I, Ia, or Ib. For a compounddescribed herein, e.g., a compound of Formula Ib, formulated into apharmaceutical composition in the form of a salt, the amount of thecompound may reflect the free base weight and not the weight of the saltform. In certain embodiments, the pharmaceutical composition of thecompound or salt of Formula I, Ia, or Ib does not include an additionalanticancer agent, e.g., a BCL-2 inhibitor or proteasome inhibitor. Incertain embodiments, the pharmaceutical composition includes anadditional anticancer agent, e.g., a BCL-2 inhibitor or proteasomeinhibitor.

A therapeutically effective amount of a compound of the disclosure,e.g., a compound or salt of Formula I, Ia, or Ib, can be expressed as mgof the compound per kg of subject body mass. In some instances, a doseof a therapeutically effective amount may be at least about 0.1 mg/kg toabout 20 mg/kg, for example, about 0.1 mg/kg, 0.2 mg/kg, 0.3 mg/kg, 0.4mg/kg, 0.5 mg/kg, 0.6 mg/kg, 0.7 mg/kg, 0.8 mg/kg, 0.9 mg/kg, 1 mg/kg,about 2 mg/kg, about 3 mg/kg, about 4 mg/kg, about 5 mg/kg, about 6mg/kg, about 7 mg/kg, about 8 mg/kg, about 9 mg/kg, about 10 mg/kg, orabout 20 mg/kg. For a compound described herein, e.g., a compound ofFormula Ib, formulated into a pharmaceutical composition in the form ofa salt, the therapeutically effective amount of the compound may reflectthe free base weight and not the weight of the salt form.

In certain embodiments, the disclosure provides a pharmaceuticalcomposition, e.g., for oral or parenteral administration, comprising aBCL-2 inhibitor, e.g., venetoclax or navitoclax. The pharmaceuticalcomposition may comprise a BCL-2 inhibitor in an amount of at leastabout 1 mg to about 1000 mg, from about 100 mg to about 1000 mg, fromabout 100 mg to about 800 mg, from about 200 mg to about 800 mg, or fromabout 300 mg to about 8000 mg. For example, a pharmaceutical compositionof the disclosure may comprise about 100 mg, about 120 mg, about 140 mg,about 160 mg, about 180 mg, about 200 mg, about 220 mg, about 240 mg,about 260 mg, about 280 mg, about 300 mg, about 320 mg, about 340 mg,about 360 mg, about 380 mg, about 400 mg, about 420 mg, about 440 mg,about 460 mg, about 480 mg, about 500 mg, about 520 mg, about 540 mg,about 560 mg, about 580 mg, about 600 mg, about 620 mg, about 640 mg,about 660 mg, about 680 mg, about 700 mg, about 720 mg, about 740 mg,about 760 mg, about 780 mg, about 800 mg, about 820 mg, about 840 mg,about 860 mg, about 880 mg, about 900 mg, about 920 mg, about 940 mg,about 960 mg, about 980 mg, or about 1000 mg of a BCL-2 inhibitor, e.g.,venetoclax or navitoclax.

In certain embodiments, the disclosure provides a pharmaceuticalcomposition, e.g., for oral or parenteral administration, comprising aproteasome inhibitor, e.g., bortezomib, marizomib, or ixazomib. Thepharmaceutical composition may comprise a proteasome inhibitor in anamount of at least about 0.5 mg to about 50 mg, from about 1 mg to about30 mg, from about 1 mg to about 20 mg, from about 1 mg to about 10 mg,or from about 1 mg to about 5 mg. For example, a pharmaceuticalcomposition of the disclosure may comprise about 0.5 mg, about 1 mg,about 2 mg, about 3 mg, about 4 mg, about 5 mg, about 6 mg, about 7 mg,about 8 mg, about 9 mg, about 10 mg, about 11 mg, about 12 mg, about 13mg, about 14 mg, about 15 mg, about 16 mg, about 17 mg, about 18 mg,about 19 mg, or about 20 mg of a proteasome inhibitor, e.g., bortezomib,marizomib, or ixazomib.

In certain embodiments, formulations of the disclosure comprise acompound or salt of Formula I, Ia, or Ib, a BCL-2 inhibitor or aproteasome inhibitor, wherein the compound or salt is about 70% to about99.99%, about 80% to about 99.9%, about 85% to about 99%, about 90% toabout 99%, about 95% to about 99%, about 97% to about 99%, about 98% toabout 99%, about 98% to about 99.9%, about 99% to about 99.99%, about99.5% to about 99.99%, about 99.6% to about 99.99%, about 99.8 to about99.99%, or about 99.9% to about 99.99% free of impurities.

In certain embodiments, a pharmaceutical composition of the disclosurecomprises both a compound or salt of Formula I, Ia, or Ib and a BCL-2inhibitor in amounts such as the ones described herein, e.g., apharmaceutical composition with 100 to 400 mg of a compound or salt ofFormula Ib and 200 to 800 mg of a BCL-2 inhibitor, e.g., venetoclax.

In certain embodiments, a pharmaceutical composition of the disclosurecomprises both a compound or salt of Formula I, Ia, or Ib and aproteasome inhibitor in amounts such as the ones described herein, e.g.,a pharmaceutical composition with 100 to 400 mg of a compound or salt ofFormula Ib and 1 to 10 mg of a proteasome inhibitor, e.g., ixazomib.

Pharmaceutical compositions disclosed herein may be in the form of aliquid formulation, a solid formulation or a combination thereof.Non-limiting examples of formulations may include a tablet, a capsule, apill, a gel, a paste, a liquid solution and a cream. In some instances,the therapeutic agent, e.g., compound or salt of Formula I, Ia, or Ib,BCL-2 inhibitor or proteasome inhibitor, may be in a crystallized form.In pharmaceutical compositions comprising two or more therapeuticagents, each agent may be crystallized separately and then combined orthey may be crystallized together. Compositions may comprise two or moretherapeutic agents in one or more physical state. For example, acomposition may be a tablet comprising one therapeutic agent in a solidformulation and another therapeutic agent or drug in a gel formulation.In certain embodiments, the composition is a single pharmaceuticalcomposition comprising a compound or salt of Formula I, Ia, or Ib in afirst physical state and a BCL-2 family inhibitor or a proteasomeinhibitor in a second physical state.

The compositions of the present disclosure may further comprise anexcipient or an additive. Excipients may include any and all solvents,coatings, chelating agents, flavorings, colorings, lubricants,disintegrants, preservatives, sweeteners, anti-foaming agents, bufferingagents, polymers, antioxidants, binders, diluents, and vehicles (orcarriers). Generally, the excipient is compatible with the therapeuticcompositions of the present disclosure.

Liquid preparations for oral administration can take the form of, forexample, solutions, syrups or suspensions, or they can be presented as adry product for reconstitution with water or other suitable vehiclesbefore use. Such liquid preparations can be prepared by conventionalapproaches with pharmaceutically acceptable additives such as suspendingagents (e.g., sorbitol syrup, methyl cellulose or hydrogenated ediblefats); emulsifying agents (e.g., lecithin or acacia); non-aqueousvehicles (e.g., almond oil, oily esters or ethyl alcohol); preservatives(e.g., methyl or propyl p-hydroxybenzoates or sorbic acid); andartificial or natural colors and/or sweeteners.

This disclosure further encompasses anhydrous compositions and dosageforms comprising an active ingredient, since water can facilitate thedegradation of some compounds. Anhydrous compositions and dosage formsof the present disclosure can be prepared using anhydrous or lowmoisture containing ingredients and low moisture or low humidityconditions. Compositions and dosage forms of the present disclosurewhich contain lactose can be made anhydrous if substantial contact withmoisture and/or humidity during manufacturing, packaging, and/or storageis expected. An anhydrous composition can be prepared and stored suchthat its anhydrous nature is maintained. Accordingly, anhydrouscompositions can be packaged using materials that prevent exposure towater such that they can be included in suitable formulary kits.Examples of suitable packaging include, but are not limited to,hermetically sealed foils, plastic, unit dose containers, blister packs,and strip packs.

An ingredient described herein can be combined in an intimate admixturewith a pharmaceutical carrier according to conventional pharmaceuticalcompounding techniques. The carrier can take a wide variety of formsdepending on the form of preparation desired for administration. Inpreparing the compositions for an oral dosage form, any of the usualpharmaceutical media can be employed as carriers, such as, for example,water, glycols, oils, alcohols, flavoring agents, preservatives,coloring agents, in the case of oral liquid preparations (such assuspensions, solutions, and elixirs) or aerosols; or carriers such asstarches, sugars, micro-crystalline cellulose, diluents, granulatingagents, lubricants, binders, and disintegrating agents can be used inthe case of oral solid preparations, in some embodiments withoutemploying the use of lactose. For example, suitable carriers includepowders, capsules, and tablets, with the solid oral preparations. Ifdesired, tablets can be coated by standard aqueous or nonaqueoustechniques.

Some examples of materials which can serve as pharmaceuticallyacceptable carriers include: (1) sugars, such as lactose, glucose andsucrose; (2) starches, such as corn starch and potato starch; (3)cellulose, and its derivatives, such as sodium carboxymethyl cellulose,ethyl cellulose and cellulose acetate; (4) powdered tragacanth; (5)malt; (6) gelatin; (7) talc; (8) excipients, such as cocoa butter andsuppository waxes; (9) oils, such as peanut oil, cottonseed oil,safflower oil, sesame oil, olive oil, corn oil and soybean oil; (10)glycols, such as propylene glycol; (11) polyols, such as glycerin,sorbitol, mannitol and polyethylene glycol; (12) esters, such as ethyloleate and ethyl laurate; (13) agar; (14) buffering agents, such asmagnesium hydroxide and aluminum hydroxide; (15) alginic acid; (16)pyrogen-free water; (17) isotonic saline; (18) Ringer's solution; (19)ethyl alcohol; (20) phosphate buffer solutions; and (21) other non-toxiccompatible substances employed in pharmaceutical formulations.

Binders suitable for use in dosage forms include, but are not limitedto, corn starch, potato starch, or other starches, gelatin, natural andsynthetic gums such as acacia, sodium alginate, alginic acid, otheralginates, powdered tragacanth, guar gum, cellulose and its derivatives(e.g., ethyl cellulose, cellulose acetate, carboxymethyl cellulosecalcium, sodium carboxymethyl cellulose), polyvinyl pyrrolidone, methylcellulose, pre-gelatinized starch, hydroxypropyl methyl cellulose,microcrystalline cellulose, and mixtures thereof.

Examples of suitable fillers for use in the compositions and dosageforms disclosed herein include, but are not limited to, talc, calciumcarbonate (e.g., granules or powder), microcrystalline cellulose,powdered cellulose, dextrates, kaolin, mannitol, silicic acid, sorbitol,starch, pre-gelatinized starch, and mixtures thereof.

When aqueous suspensions and/or elixirs are desired for oraladministration, the active ingredient therein can be combined withvarious sweetening or flavoring agents, coloring matter or dyes and, ifso desired, emulsifying and/or suspending agents, together with suchdiluents as water, ethanol, propylene glycol, glycerin and variouscombinations thereof.

In one embodiment, the composition can include a solubilizer to ensuregood solubilization and/or dissolution of the compound of the presentdisclosure and to minimize precipitation of the compound of the presentdisclosure. This can be especially important for compositions fornon-oral use, e.g., compositions for injection. A solubilizer can alsobe added to increase the solubility of the hydrophilic drug and/or othercomponents, such as surfactants, or to maintain the composition as astable or homogeneous solution or dispersion.

Pharmaceutical compositions described herein may be suitable for oraladministration to a subject in need thereof. In some cases, slow releaseformulations for oral administration may be prepared in order to achievea controlled release of the active agent in contact with the body fluidsin the gastrointestinal tract, and to provide a substantial constant andeffective level of the active agent in the blood plasma. The crystalform may be embedded for this purpose in a polymer matrix of abiological degradable polymer, a water-soluble polymer or a mixture ofboth, and optionally suitable surfactants. Embedding can mean in thiscontext the incorporation of micro-particles in a matrix of polymers.Controlled release formulations are also obtained through encapsulationof dispersed micro-particles or emulsified micro-droplets via knowndispersion or emulsion coating technologies.

In some embodiments, the compositions can be formulated in a foodcomposition. For example, the compositions can be a beverage or otherliquids, solid food, semi-solid food, with or without a food carrier.For example, the compositions can include a black tea supplemented withany of the compositions described herein. The composition can be a dairyproduct supplemented any of the compositions described herein. In someembodiments, the compositions can be formulated in a food composition.For example, the compositions can comprise a beverage, solid food,semi-solid food, or a food carrier.

In certain embodiments, the pharmaceutical formulations can be in a formsuitable for parenteral injection as a sterile suspension, solution, oremulsion in oily or aqueous vehicles, and can contain formulation agentssuch as suspending, stabilizing, and/or dispersing agents.Pharmaceutical formulations for parenteral administration include, forexample, aqueous solutions of the active compounds in water-solubleform. Suspensions of the active compounds can be prepared, for example,as oily injection suspensions. Suitable lipophilic solvents or vehiclesinclude fatty oils such as sesame oil, or synthetic fatty acid esters,such as ethyl oleate, isopropyl palmitate, or medium chaintriglycerides, or liposomes. In preferred embodiments, a formulation forparenteral administration is an aqueous suspension.

The compound described herein may be present in a composition within arange of concentrations, the range being defined by an upper and lowervalue selected from any of the preceding concentrations. For example,the compound or salt of the disclosure may be present in the formulationat a concentration of from about 1 nM to about 100 mM, about 10 nM toabout 10 mM, about 100 nM to about 1 mM, about 500 nM to about 1 mM,about 1 mM to about 50 mM, about 10 mM to about 40 mM, about 20 mM toabout 35 mM, or about 20 mM to about 30 mM.

Methods for the preparation of compositions comprising the compoundsdescribed herein can include formulating the compounds with one or moreinert, pharmaceutically-acceptable excipients. Liquid compositionsinclude, for example, solutions in which a compound is dissolved,emulsions comprising a compound, or a solution containing liposomes,micelles, or nanoparticles comprising a compound as disclosed herein.These compositions can also contain minor amounts of nontoxic, auxiliarysubstances, such as wetting or emulsifying agents, pH buffering agents,and other pharmaceutically-acceptable additives.

Formulations for injection can be presented in unit dosage form, forexample, in ampoules or in multi-dose containers, with an addedpreservative. The compositions can take such forms as suspensions,solutions or emulsions in oily or aqueous vehicles, and can containformulatory agents such as suspending, stabilizing and/or dispersingagents. The compositions can be presented in unit-dose or multi-dosecontainers, for example sealed ampoules and vials, and can be stored inpowder form or in a freeze-dried (lyophilized) condition requiring onlythe addition of the sterile liquid carrier, for example, saline orsterile pyrogen-free water, immediately prior to use. Extemporaneousinjection solutions and suspensions can be prepared from sterilepowders, granules and tablets of the kind previously described.

Pharmaceutical formulations for parenteral administration includeaqueous and non-aqueous (oily) sterile injection solutions of the activecompounds which can contain antioxidants, buffers, bacteriostats andsolutes which render the formulation isotonic with the blood of theintended recipient; and aqueous and non-aqueous sterile suspensionswhich can include suspending agents and thickening agents. Suitablelipophilic solvents or vehicles include fatty oils such as sesame oil,or synthetic fatty acid esters, such as ethyl oleate or triglycerides,or liposomes.

A composition described herein, e.g., a pharmaceutical composition of acompound or salt of Formula I, Ia, or Ib, or a BCL-2 inhibitor or aproteasome inhibitor or a co-formulation of a compound of Formula I, Ia,or Ib with a BCL-2 inhibitor or proteasome inhibitor, can beadministered once or more than once each day. The composition may beadministered serially (e.g., taken every day without a break for theduration of the treatment regimen). In some cases, the treatment regimecan be less than a week, a week, two weeks, three weeks, a month, orgreater than a month. In some cases, a composition of the disclosure isadministered over a period of at least 12 weeks. In other cases, thecomposition is administered for a day, at least two consecutive days, atleast three consecutive days, at least four consecutive days, at leastfive consecutive days, at least six consecutive days, at least sevenconsecutive days, at least eight consecutive days, at least nineconsecutive days, at least ten consecutive days, or at least greaterthan ten consecutive days. In some cases, a therapeutically effectiveamount can be administered one time per week, two times per week, threetimes per week, four times per week, five times per week, six times perweek, seven times per week, eight times per week, nine times per week,10 times per week, 11 times per week, 12 times per week, 13 times perweek, 14 times per week, 15 times per week, 16 times per week, 17 timesper week, 18 times per week, 19 times per week, 20 times per week, 25times per week, 30 times per week, 35 times per week, 40 times per week,or greater than 40 times per week. In some cases, a therapeuticallyeffective amount can be administered one time per day, two times perday, three times per day, four times per day, five times per day, sixtimes per day, seven times per day, eight times per day, nine times perday, 10 times per day, or greater than 10 times per day. In some cases,the composition is administered at least twice a day. In further cases,the composition is administered at least every hour, at least every twohours, at least every three hours, at least every four hours, at leastevery five hours, at least every six hours, at least every seven hours,at least every eight hours, at least every nine hours, at least every 10hours, at least every 11 hours, at least every 12 hours, at least every13 hours, at least every 14 hours, at least every 15 hours, at leastevery 16 hours, at least every 17 hours, at least every 18 hours, atleast every 19 hours, at least every 20 hours, at least every 21 hours,at least every 22 hours, at least every 23 hours, or at least every day.

Pharmaceutical compositions of the disclosure can be administered eitheracutely or chronically. Pharmaceutical compositions of the invention canbe administered as a single treatment or as a course of treatment.Treatments can be administered once per day, twice per day, three timesper day, in the morning, in the evening, before sleeping, orcontinuously throughout the day. Treatments can be applied every day,every other day, every three days, twice weekly, once weekly, everyother week, monthly, every six weeks, every other month, every threemonths, every six months, annually, every other year, every 5 years, oras required.

In certain embodiments, the dose of drug being administered may betemporarily reduced or temporarily suspended for a certain length oftime. In certain embodiments, the patient will have a drug holidaywherein the patient does not receive the drug or receives a reducedamount of the drug for a period of time. A drug holiday can be, forexample, between 2 days and 1 year, including by way of example only, 2days, 3 days, 4 days, 5 days, 6 days, 7 days, 10 days, 12 days, 15 days,20 days, 28 days, or more than 28 days. A drug holiday may be for about1 month, about 2 months, about 3 months, about 4 months, about 5 months,about 6 months, about 7 months, about 8 months, about 9 months, about 10months, about 11 months or about 12 months. The dose reduction during adrug holiday can be, for example, by 10%-100% of the originaladministered dose, including by way of example only 10%, 15%, 20%, 25%,30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%,and 100%. For further examples the dose reduction can be between 10% and100%, between 20% and 80%, between 30% and 70%, between 50% and 90%,between 80% and 100% or between 90% and 100%.

Once improvement of the patient's conditions has occurred, a maintenancedose can be administered if necessary. Subsequently, the dosage or thefrequency of administration, or both, can be reduced, as a function ofthe symptoms, to a level at which the improved disease, disorder orcondition can be retained.

Additional methods for administering the formulations described hereininclude, for example, limited to delivery via enteral routes includingoral, gastric or duodenal feeding tube, rectal suppository, rectalenema, parenteral routes, injection, infusion, intraarterial,intracardiac, intradermal, intraduodenal, intramedullary, intramuscular,intraosseous, intraperitoneal, intrathecal, intravascular, intravenous,intravitreal, intracameral, epidural, subcutaneous, inhalational,transdermal, transmucosal, sublingual, buccal, topical, epicutaneous,dermal, enemaear drops, intranasal, and vaginal administration. Thecompounds described herein can be administered locally to the area inneed of treatment, by for example, local infusion during surgery,topical application such as creams or ointments, injection, catheter, orimplant. The administration can also be by direct injection at the siteof a diseased tissue or organ.

The length of the period of administration and/or the dosing amounts canbe determined by a physician or any other type of clinician. Thephysician or clinician can observe the subject's response to theadministered compositions and adjust the dosing based on the subject'sperformance. For example, dosing for subjects that show reduced effectsin energy regulation can be increased to achieve desired results.

In some embodiments, the combination therapies described herein can beadministered together at the same time in the same route, oradministered separately. In some embodiments, the components in thecompositions can be administered using the same or differentadministration routes.

In some embodiment, the disclosure also provides for methods ofmanufacturing the compositions described herein. In some embodiments,the manufacture of a composition described herein comprises mixing orcombining two or more components.

In some embodiments, the compositions can be combined or mixed with apharmaceutically active or therapeutic agent, a carrier, and/or anexcipient. Examples of such components are described herein. Thecombined compositions can be formed into a unit dosage as tablets,capsules, gel capsules, slow-release tablets, or the like.

In some embodiments, the composition is prepared such that a solidcomposition containing a substantially homogeneous mixture of the one ormore components is achieved, such that the one or more components aredispersed evenly throughout the composition so that the composition canbe readily subdivided into equally effective unit dosage forms such astablets, pills and capsules.

A unit dose may be packaged into a container to be transferred to theuser. A unit dose may be packaged in a tube, a jar, a box, a vial, abag, a tray, a drum, a bottle, a syringe, or a can.

Another aspect of the disclosure provides for achieving desired effectsin one or more subjects after administration of a combinationcomposition described herein for a specified time period. For example,the beneficial effects of the compositions described herein can beobserved after administration of the compositions to the subject for 1,2, 3, 4, 6, 8, 10, 12, 24, or 52 weeks.

In certain embodiments, the combination therapies described herein maybe administered by a combination treatment regimen. A combinationtreatment regimen can encompass treatment regimens in whichadministration of a compound described herein, or a pharmaceuticallyacceptable salt thereof, is initiated prior to, during, or aftertreatment with a second agent described herein, and continues until anytime during treatment with the second agent or after termination oftreatment with the second agent. The disclosure also includes treatmentsin which a compound described herein, or a pharmaceutically acceptablesalt thereof, and the second agent being used in combination areadministered simultaneously or at different times and/or at decreasingor increasing intervals during the treatment period. Combinationtreatment further includes periodic treatments that start and stop atvarious times to assist with the clinical management of the patient.

In certain embodiments, the combination therapy can provide atherapeutic advantage in view of the differential toxicity associatedwith the two treatment modalities. For example, treatment with CDKinhibitors such as those described herein can lead to a particulartoxicity that is not seen with the anticancer agent, e.g., BCL-2inhibitor or proteasome inhibitor, and vice versa. As such, thisdifferential toxicity can permit each treatment to be administered at adose at which said toxicities do not exist or are minimal, such thattogether the combination therapy provides a therapeutic dose whileavoiding the toxicities of each of the constituents of the combinationagents. Furthermore, when the therapeutic effects achieved as a resultof the combination treatment are synergistic, the doses of each of theagents can be reduced even further, thus lowering the associatedtoxicities to an even greater extent.

The compounds described herein or the pharmaceutically acceptable saltsthereof, as well as combination therapies, may be administered before,during or after the occurrence of a disease or condition, and the timingof administering the composition containing a compound varies. Thecompounds described herein can be used as a prophylactic and may beadministered continuously to subjects with a propensity to developconditions or diseases in order to prevent the occurrence of the diseaseor condition. The compounds described herein and compositions thereofmay be administered to a subject during or as soon as possible after theonset of the symptoms. A compound described herein may be administeredas soon as is practicable after the onset of a disease or condition isdetected or suspected, and for a length of time necessary for thetreatment of the disease.

EXAMPLES

The following examples are given for the purpose of illustrating variousembodiments of the invention and are not meant to limit the presentinvention in any fashion. The present examples, along with the methodsdescribed herein are presently representative of preferred embodiments,are exemplary, and are not intended as limitations on the scope of theinvention. Changes therein and other uses which are encompassed withinthe spirit of the invention as defined by the scope of the claims willoccur to those skilled in the art.

For all in vitro drug mechanism-of-action studies, the materials andmethods outlined below were used.

Cell lines shown in Table 1, adherent or suspension, were treated in6-well dishes or T10 flasks respectively, seeded at one million per mldensity prior to treatment in total volumes of 3-10 ml. All agents usedfor in vitro cell treatments were formulated in water/DMSO to achieverequired concentrations (as stated in respective figure labels/legends)with DMSO concentrations not exceeding 0.01%. Drug sources are indicatedin table under ‘CIVO microinjection studies’. Cells were harvested atexperimental time points: 2, 6, or 24 hours post treatment as indicatedin respective figures, first by washing cells twice in ice-cold PBS in15 ml falcon tubes, centrifugation at 1000 rpm for 5 mins followed bylysis of the respective cell pellets as described in section below.Note: Drug resistant NUDHL1 Dox 1000 nM and Toledo Dox 1000 nM celllines were generated at Presage by culturing parental lines in mediawith stepwise increments in Doxorubicin concentration over severalpassages. These cells were used to generate xenografts when resistanceto 1 uM Dox was achieved.

For all Western Blot Analysis studies, the materials and methodsoutlined below were used.

Protein lysates were prepared by adding ice-cold 1× RIPA Buffer (from10×, Millipore) with 1×-3× Halt protease and phosphatase inhibitorcocktail (100×, Pierce) to cell pellets, followed by pulse sonication(˜3-5 secs) three times. Lysates were cleared by centrifugation at 14Krpm for 15-20 mins and the supernatant (lysate) was collected forfurther analysis. To prepare lysates from cryopreserved xenograft tumortissue, tissue was first homogenized using a hand-held tissuehomogenizer (Cole Palmer) in above lysis buffer followed by pulsesonication and centrifugation as described above. Above steps werecarried out under ice cold conditions. Protein samples were quantifiedusing Bradford assay (Biorad) at 1:10 or higher dilutions usingappropriate blank controls with 1× RIPA. 20-25 micrograms of proteinfrom each sample were subject to SDS-PAGE using BOLT 4-12% Bis-Tris gels(Thermofisher) and transferred to either 0.45 or 0.2 micron pore sizemembranes (as applicable based on protein molecular size), and eithernitrocellulose (NC) or pvdf (Thermofisher) using manufacturer'sprotocol, blocked for one hour in 5% milk at room temperature, probedwith primary antibodies (Table 2) at 4 deg C. overnight andcorresponding HRP-conjugated secondary antibodies (JacksonImmunoresearch, 1:10,000-1:20,000 dilutions) for 1 hour at roomtemperature. Three PBS/0.01% Tween-20 washes (5-10 mins each) werecarried out after each antibody incubation. ECL chemiluminescentsubstrate (Pierce or GE) and autoradiographic film (Thermofisher) wasused for detection of protein signals.

For all in vivo studies, the materials and methods outlined below wereused.

All work in mice was approved by IACUC Board of Presage Biosciences,Seattle, Wash. All relevant procedures were performed under anesthesiaand all efforts were made to minimize pain and suffering. None of themice contributing to this study became ill or died prior to experimentalendpoints and all mice receiving drug treatment as described below,underwent routine health monitoring and were humanely euthanized at theend of the experiments. Subcutaneous flank xenografts were generatedusing the following cell lines/mouse strains tabulated below (Table 3).200 ul cell suspension in a 1:1 ratio with matrigel (Corning) wasinjected into the right flank using 1 ml syringes (BD 309659) and 27 Gneedles (BD 305109).

For all CIVO microinjection studies, the materials and methods outlinedbelow were used.

Mice were enrolled into CIVO drug studies when the tumor volume reachedapproximately 1000 mm³. Microinjection studies were performed using theCIVO device as previously described (Klinghoffer et al, ScienceTranslational Medicine 2015) The device was configured with 6 injectionneedles set for a 6 mm injection length and a total volume delivery of 3μl. A fluorescent tracking marker (FTM) was added to each drug reservoirin vehicle for delivery along with each drug or drug combination. Allmicro-doses were equivalent to or lower than what would be allowed underFDA guidelines for Exploratory IND (Investigational New Drug) studiesand by solubility of drug into vehicle. Total amounts of agents injectedare tabulated below in Table 4.

For all systemic drug efficacy studies, the materials and methodsoutlined below were used.

Mice were enrolled for study when tumors reached an average volume of150-200 mm³. Tumor volume was calculated as V=length×width×height, allthree dimensions measured using digital calipers along with body weight.Animals were removed from the study when—any one of the three measureddimensions of the tumor exceeding 2 cm, volume exceeding 2500 mm³,ulceration or body weight loss greater than 20%.

Tumor Growth Inhibition % (TGI) is defined as:

$\frac{\begin{matrix}{\left( {{V_{final}({vehicle})} - {V_{initial}({vehicle})}} \right) -} \\\left( {{V_{final}({treatment})} - {V_{initial}({treatment})}} \right)\end{matrix}}{\left( {{V_{final}({vehicle})} - {V_{initial}({vehicle})}} \right)} \times 100$

where measurements are averaged across tumors in respective arms.Wilcoxon Rank Sum or Mann-Whitney test was used as the statistical testto determine differences between treatment arms.

Drug doses, routes of administration, schedules and formulationprotocols are detailed below. For oral gavage, Instech, Ref: FTP-20-38feeding tubes and for IV, BD insulin syringes were used.

DLBCL Studies

-   -   RIVA: Voruciclib 200 mpk (PO) 6 on/1 off and ABT 199 (PO) 1 mpk        2×/week for 4 weeks    -   SUDHL4: Voruciclib 200 mpk (PO) 6 on/1 off and ABT 199 (PO) 25        mpk 6 on/1 off for 4 weeks    -   U2932: Voruciclib 200 mpk (PO) 6 on/1 off and ABT 199 (PO) 10        mpk 2×/week for 4 weeks

AML Studies

-   -   SKM1: Voruciclib 200 mpk (PO) 6 on/1 off+ABT 199 (PO) 10 mpk 6        on/1 off for 3 weeks

TNBC Studies

-   -   HCC1187: Voruciclib 200 mpk (PO) 5 on/2 off+Ixazomib/MLN2238        (PO) 3 mpk Days 3. 6. 10, 13 for 2 weeks    -   HCC1187: Palbociclib 120 mpk (PO) QD×14+Bortezomib 0.42 mpk        (IV), Days 1, 4, 8, 11 for 2 weeks    -   HCC1187: Voruciclib 200 mpk (PO)+Bortezomib 0.42 mpk (IV), Days        1, 4, 8 11 for 2 weeks

Example 1 Voruciclib is a Potent Inhibitor of Cyclin-Dependent Kinase-9(CDK9)

Voruciclib is thought to be a potent CDK4/6 inhibitor. The activity ofvoruciclib on 38 different kinases was tested in comparison toflavopiradol, a known CDK9 inhibitor. FIG. 1 depicts the structures ofvoruciclib and flavopiridol, as well as a comparison of the inhibitionprofiles of voruciclib and flavopiradol. FIG. 1 demonstrates that,similar to flavopiradol, voruciclib is a potent inhibitor of CDK9(K_(i)<10 nM). However, voruciclib is more specific for CDKs thanflavopiradol. FIG. 1 shows that flavopiradol is a potent inhibitor ofthe serine/threonine protein kinases MAK and ICK, whereas voruciclibdoes not show potent inhibition of either MAK or ICK. MAK and ICK areassociated with gut epithelial cells and may account for the severediarrhea associated with flavopiradol treatment. Voruciclib, on theother hand, may exhibit fewer gastrointestinal side effects. DiscoveRxand Thermofisher screens: Percent Inhibition of kinase activity for 468kinase assays of the DiscoveRx ScanMax kinase panel and 414 kinaseassays of the ThermoFisher SelectScreen kinase panel at 10 uM and 50 nM.Test Article is pre-weighted at Presage into silanized amber screw capvials and shipped to DiscoveRx and Reaction Biology. For ThermoFisher,the compound is weighed in and dissolved at 10 mM DMSO prior to shipmentat ambient conditions. On receipt, vials are stored at −20 C until used.Powder is dissolved in DMSO at 10 mM.

FIG. 2 shows single digit nM potency of voruciclib for CDKs 1, 4, 6, and9 with strongest inhibition of CDK9. Reaction Biology profiling:Objective of the study was to determine the rank order of sensitivity of48 kinases to voruciclib hydrochloride. The testing facility wasReaction Biology Corp. Kinase activity was measured using a filterbinding assay with radioactive γ-33P-ATP as phosphate donor. The ATPconcentration was near the Km for respective kinase. For each kinase, anIC50 value were calculated from a 10-point concentration curve of thetest article and converted to Ki values. The 48 kinases studied here hadbeen identified in previous screening experiments as the most promisingtarget candidates.

Example 2 Voruciclib Exhibits a Synergistic Effect in Combination withthe Bcl-2 Family Inhibitor, Venetoclax (ABT-199)

NU-DHL-1 diffuse large B-cell lymphoma (DLBCL) cells were treated withvehicle, voruciclib alone, venetoclax (ABT-199) alone, or a combinationof voruciclib and venetoclax. FIGS. 3A-3D show NU-DHL-1 cells stainedfor the anti-apoptotic protein, induced myeloid leukemia celldifferentiation protein Mcl-1 (MCL-1) (shown in red). FIG. 3A showscells that were treated with vehicle only. In FIG. 3B, Voruciclib alonedecreased the expression of MCL-1, shown by the increase in darker areasoutlines in outline 301. Venetoclax alone increased the expression ofMCL-1, as shown in the light colored areas within outline 302 of FIG.3C. The combination of voruciclib and venetoclax led to a markeddecrease in MCL-1 expression, shown in FIG. 3D, shown in the dark areasof outline 303.

FIGS. 4A-4D demonstrates a correlation between MCL-1 suppression with asynergistic induction of apoptosis. NU-DHL-1 diffuse large B-celllymphoma (DLBCL) cells were treated with vehicle, voruciclib alone,venetoclax (ABT-199) alone, or a combination of voruciclib andvenetoclax. The cells were stained for cleaved caspase-3 (CC3) (shown inred) as a marker of apoptosis. FIG. 4A shows cells that were treatedwith vehicle only. Voruciclib alone, shown in FIG. 4B, showed a slightincreased the expression of CC3, as shown by the light colored areaswithin outline 401. Similarly, venetoclax alone, shown in FIG. 4C,showed a slight increased the expression of CC3, as shown by the lightcolored areas within outline 402. The combination of voruciclib andvenetoclax in FIG. 4D showed synergistically increased expression ofCC3, as shown in the large amount of light colored area within outline403, suggesting an increased induction of apoptosis in the treatedcells.

FIGS. 6A-6E demonstrate that the synergistic effect of voruciclib andvenetoclax on apoptosis is reproducible across multiple DLBCL models.Briefly, RIVA, Toledo, Toledo/Dox1000, NUDHL, and NUDHL/Dox1000 cellswere treated with voruciclib alone, venetoclax alone or voruciclib andvenetoclax in combination, as shown in FIG. 6A, FIG. 6B, FIG. 6C, FIG.6D, and FIG. 6E, respectively. Voruciclib alone and venetoclax alone hada slight effect on CC3 expression, however, the combination ofvoruciclib and venetoclax demonstrated a synergistic induction of CC3expression across all five DLBCL models.

FIG. 7 depicts a proposed model of this synergistic effect. Voruciclib,through inhibition of MCL-1 via CDK9, may shift cells to apoptosis whencombined with Bcl-2 inhibition.

Example 3 Voruciclib Exhibits a Synergistic Effect in Combination withthe Bcl-2 Family Inhibitor, Navitoclax

Ramos Burkitt's lymphoma cells were treated with vehicle, voruciclibalone, navitoclax (ABT-263) alone, or a combination of voruciclib andnavitoclax. FIGS. 5A-5D show Ramos Burkitt's lymphoma cells stained forcleaved caspase-3 (CC3) (shown in red) as a marker of apoptosis. FIG. 5Ashows cells that were treated with vehicle only. Voruciclib alone, asshown in FIG. 5B, showed slight increase in the expression of CC3, asshown within the light areas of outline 501. Similarly, Venetoclaxalone, as shown in FIG. 5C, showed slight increase in the expression ofCC3, as shown within the light areas of outline 502. The combination ofvoruciclib and navitoclax showed a marked increase in the expression ofCC3, as shown in the large amount of light colored area within outline503 of FIG. 5D, suggesting an increased induction of apoptosis in thetreated cells.

Example 4 Voruciclib Exhibits a Synergistic Effect in Combination withProteasome Inhibitors

A drug screen was performed to test multiple drugs simultaneously. FIG.8A shows a list of various compounds that were injected into the HCC1187triple negative breast cancer (TNBC) xenograft model. FIG. 8B shows themean % change in MCL-1 induction in xenografts. The proteasomeinhibitor, bortezomib showed an increase in the expression of MCL-1.FIG. 8C demonstrates xenografts that were treated with vehicle. FIG. 8Dshows xenografts that were treated with bortezomib and stained for MCL-1expression (shown in red). Bortezomib markedly increased the expressionof MCL-1 in these cells, as shown by the increase of light colored areaswithin outline 801.

Example 5 Voruciclib Exhibits a Synergistic Effect in Combination withthe Proteasome Inhibitor, Marizomib

NudHL1 DLBCL xenografts were treated for 6 hours with vehicle,voruciclib alone, marizomib alone, or voruciclib and marizomib incombination. FIGS. 9A-9D depict cells that were treated as above andstained for cleaved caspase-3 (CC3) as a marker of apoptosis (shown inred). FIG. 9A shows cells that were treated with vehicle only. Cellstreated with voruciclib alone, as shown in FIG. 9B, had little to noincrease in CC3 expression, as shown by the small amount of lightcolored area within outline 901. Similarly, cells treated with marizomibalone, as shown in FIG. 9C, had little to no increase in CC3 expression,as shown by the small amount of light colored area within outline 902.Cells treated with combination voruciclib and marizomib showed a markedincrease in CC3 expression, as shown by the large amount of lightcolored area within outline 903 of FIG. 9D, suggesting a synergisticeffect on induction of apoptosis in these cells.

Example 6 Voruciclib Exhibits a Synergistic Effect in Combination withthe Proteasome Inhibitor, Bortezomib

Briefly, NudHL1 DLBCL xenografts were treated for 6 hours with vehicle,voruciclib alone, bortezomib alone, or voruciclib and bortezomib incombination. FIGS. 10A-10D depict cells that were treated as above andstained for cleaved caspase-3 (CC3) as a marker of apoptosis (shown inred).

FIG. 10A shows cells that were treated with vehicle only. Cells treatedwith voruciclib alone, as shown in FIG. 10B, had little to no increasein CC3 expression. Similarly, cells treated with bortezomib alone, asshown in FIG. 10C, had little to no increase in CC3 expression, as shownby the small amount of light colored area within outline 1001. Cellstreated with combination voruciclib and bortezomib showed a markedincrease in CC3 expression, as shown by the large amount of lightcolored area within outline 1002 of FIG. 10D, suggesting a synergisticeffect on induction of apoptosis in these cells.

FIG. 11 depicts a similar experiment performed on the HCC1187 triplenegative breast cancer (TNBC) xenograft model. Briefly, cells weretreated with vehicle, voruciclib alone, bortezomib alone, or vorucicliband bortezomib in combination. Cells were then stained for CC3expression (shown in red) as a marker of apoptosis. FIG. 11A shows cellsthat were treated with vehicle only. Cells treated with voruciclibalone, as shown in FIG. 11B, had little to no increase in CC3expression. Similarly, cells treated with bortezomib alone, as shown inFIG. 11C, had little to no increase in CC3 expression. Cells treatedwith combination voruciclib and bortezomib showed a marked increase inCC3 expression, as shown by the large amount of light colored areawithin outline 1101 of FIG. 11D, suggesting a synergistic effect oninduction of apoptosis in these cells.

HCC1187 triple negative breast cancer (TNBC) xenograft mouse models weretreated with vehicle, voruciclib (200 mg/kg), bortezomib (0.42 mg/kg),or voruciclib (200 mg/kg) and bortezomib (0.42 mg/kg) in combination.Tumor volume was measured in each mouse over time and the results areshown in FIGS. 12A-12D. Mice treated with a combination of vorucicliband bortezomib (FIG. 12D) showed a marked reduction in normalized tumorvolume over time as compared to mice treated with voruciclib alone (FIG.12B) or bortezomib alone (FIG. 12C). FIG. 12E shows a Western blot forMLC-1 protein expression. Cells treated with voruciclib reduced MCL-1protein expression as compared to an untreated sample whereas cellstreated with bortezomib increased MCL-1 protein expression as comparedto the untreated sample. Further, voruciclib decreased thebortezomib-induced increase in MCL-1 expression.

FIG. 13 demonstrates that body weight of mice was unaffected bytreatment with voruciclib alone, bortezomib alone or voruciclib andbortezomib in combination. This data may suggest that combinationtherapy of voruciclib and bortezomib may exhibit little to no toxicity.

Further, voruciclib abrogates bortezomib-induced upregulation ofpro-survival proteins, MCL-1 and E3 ubiquitin-protein ligase XIAP. FIG.15A depicts a proposed model of voruciclib inhibition of CDK9. FIG. 15Bshows MCL-1 and XIAP protein levels in cells treated with voruciclibalone, bortezomib alone, or voruciclib and bortezomib in combination.The Western blot demonstrates that voruciclib diminishes thebortezomib-induced upregulation of both MCL-1 and XIAP proteins in thesecells.

Example 7 Palbociclib, a CDK 4/6 Inhibitor, does not Exhibit aSynergistic Effect in Combination with Bortezomib

HCC1187 triple negative breast cancer (TNBC) xenograft mouse models weretreated with vehicle, palbociclib (120 mg/kg), bortezomib (0.42 mg/kg),or palbociclib (120 mg/kg) and bortezomib (0.42 mg/kg) in combination.Tumor volume was measured in each mouse over time and the results areshown in FIG. 14. Unlike voruciclib, palbociclib did not exhibit asynergistic effect on tumor volume in combination with bortezomib.Palbociclib is a specific CDK4/6 inhibitor whereas voruciclib targetsCDK9. Thus, this difference may be CDK9-specific.

Example 8 The Endoplasmic Reticulum (ER) Stress Response Pathway mayPlay a Role in the Synergistic Effects of Voruciclib in Combination withBortezomib

FIG. 16A shows a tissue section that was treated with bortezomib.Bortezomib induces apoptosis in some cells, while some cells appear tobe resistant to bortezomib treatment. The cells were stained for the ERchaperone protein, GRP178/BiP. FIG. 16B demonstrates that the cellsresistant to apoptosis by bortezomib express GRP178/BiP.

Next, HCC1187 triple negative breast cancer cells were treated withvoruciclib alone, bortezomib alone, tunicamycin alone (an ER stressinducer), or in combination. Tunicamycin alone strongly induced X-boxbinding protein 1 (XBP1), an ER stress protein of the IRE1α arm of theER stress pathway, at 6 hours. Further, tunicamycin and bortezomibstrongly induced XBP1 expression at 24 hours (FIG. 18B). Combination ofvoruciclib with either bortezomib or tunicamycin diminished XBP1upregulation. These results suggest that voruciclib may inhibit theIRE1α arm of the ER stress pathway (FIG. 18B). Voruciclib also decreasedbortezomib-induced transcriptional induction of XBP1 (FIGS. 19A-19B).HCC1187 cells were treated with Voruciclib (1.5 uM), Bortezomib (10 nM),STF083010 IRE1a endoribonuclease activity inhibitor (Millipore) (60 uM)or Tunicamycin (Sigma) (100 nM) for 4 hours. mRNA was harvested usingQiagen RNAeasy Kit, quantified using a Nanodrop spectrophotometer. 1 ugof mRNA from each condition was used to generate cDNA using the HighCapacity cDNA Reverse Transcription Kit (Applied Biosystems) andmanufacturer's protocol. 100 ng cDNA was used to conduct a PCR assay toamplify XBP1 u or splice forms (Thermofisher PCR Supermix)—94 deg C. for2 mins, (94 deg C. for 15 secs, 55 deg C. for 30 secs, 72 deg C. for 1min)×30 cycles, 72 deg C. for 7 mins, hold at 4 deg C. XBP1 spiced andunspliced variants were detected.

Example 9 Voruciclib Exhibits a Synergistic Effect in Combination withthe Proteasome Inhibitor, Ixazomib

FIGS. 20A-20D depicts the HCC1187 triple negative breast cancer (TNBC)xenograft model. Briefly, cells were treated with vehicle, voruciclibalone, ixazomib alone, or voruciclib and ixazomib in combination. Cellswere then stained for CC3 expression (shown in red) as a marker ofapoptosis.

FIG. 20A shows cells that were treated with vehicle only. Cells treatedwith voruciclib alone, as shown in FIG. 20B, had little to no increasein CC3 expression, as shown by the small amount of light colored areawithin outline 2001. Similarly, cells treated with ixazomib alone, asshown in FIG. 20C, had little to no increase in CC3 expression, as shownby the small amount of light colored area within outline 2002. Cellstreated with combination voruciclib and ixazomib showed a markedincrease in CC3 expression, as shown by the large amount of lightcolored area within outline 2003 of FIG. 20D, suggesting a synergisticeffect on induction of apoptosis in these cells.

Various CDK inhibitors were tested in combination with ixazomib, asshown in FIGS. 21A and 21B. Only voruciclib demonstrated a synergisticeffect on apoptosis of tumor cells in combination with ixazomib, asshown by the light colored areas within outlines 2101 and 2102. Vehicleonly, ixazomib only, palbociclib and ixazomib, dinaciclib and ixazomib,and flavopiradol and ixazomib all did not exhibit the synergistic effecton apoptosis of tumor cells as the combination of voruciclib andixazomib did.

Example 10 Voruciclib Exhibits a Synergistic Effect in Combination withthe Venetoclax

FIGS. 22A-B illustrate the synergy from voruciclib and venetoclax in theSU-DHL-4 model of DLBCL. Cleaved PARP (cPARP) may be used as a biomarkerof apoptosis. To assess combination efficacy of the two agents ofvoruciclib and venetoclax together, low doses of each (150 nM vorucicliband 20 nM for venetoclax) were examined as single agents or as acombination in the SUDHL4 model of DLBCL. Cells were exposed to drug for24 h prior to cell lysis and examination by Western Blot usingantibodies specific for the cleaved form of PARP, MCL-1, C-MYC, and Betaactin (as a protein loading control) of FIG. 22A and BCL2 as anadditional protein loading control in FIG. 22B. FIG. 22A illustratesthat voruciclib suppresses MCL1 at 1.5 uM in DLBCL model.

FIG. 22B utilizes a 0.15 uM concentration of voruciclib, which is 1/10that is shown to effectively induce apoptosis as a single agent. Thislevel of voruciclib maintains some efficacy for repressing MCL-1expression (compare lanes 1 & 2 of FIG. 22B) while cell exposure tovenetoclax induces MCL-1 expression (compare lanes 1 & 3 of FIG. 22B).Low vorucicilib exposure also represses venetoclax-induced MCL-1(compare lanes 3 & 4 of FIG. 22B).

FIGS. 23A-C illustrates the synergy from voruciclib and venetoclax inthe SU-DHL-4 model, the OCI Ly10 model, and the U2932 model of DLBCL.Cleaved PARP (cPARP) may be used as a biomarker of apoptosis. To assesscombination efficacy of the two agents of voruciclib and venetoclaxtogether, low doses of each were examined as single agents or as acombination in the three models of DLBCL. Cells were exposed to drug for24 h prior to cell lysis and examination by Western Blot usingantibodies specific for the cleaved form of PARP and Beta actin (as aprotein loading control).

FIG. 23A illustrates that the combination of 0.15 uM voruciclib and 20nM of venetoclax exhibits synergy to induce apoptosis in the SUDHL4model of DLBCL that neither compound induces apoptosis as a singleagent. FIG. 23B illustrates that the combination of 5 uM voruciclib and50 nM of venetoclax exhibits synergy to induce apoptosis in the OCI Ly10model of DLBCL where neither compound significantly induces apoptosis asa single agent. FIG. 23C illustrates that the combination of 5 uMvoruciclib and 50 nM of venetoclax exhibits synergy to induce apoptosisin the U2932 model of DLBCL where the combination of the two compoundsis clearly greater than just the addition of each compound as a singleagent.

Example 11 Voruciclib Suppresses MCL1 Expression in DLBCL XenograftTumors in Mice

FIG. 24 illustrates that voruciclib suppresses MCL1 expression in DLBCLxenograft tumors in mice. Mice harboring DLBCL xenograft tumors(OCILY10) were dosed with voruciclib or vehicle control by oral gavagedaily for 5 days and tumors were harvested for analysis of MCL-1expression 4 h post the final dose. Tumor samples were cut in half, withone half solubilized in lysis buffer for western blot analysis and theother half fixed in formalin and prepared for immunohistochemistry.Western blots of tumor lysates from 3 individual mice per treatmentgroup with antibodies specific for MCL-1 or Beta actin (protein loadingcontrol). The graph illustrates that voruciclib as a single agentsuppressed MCL1 expression in DLBCL xenograft tumors at 100 mpk.

Example 12 Voruciclib and Venetoclax Impede Growth of DLBCL XenograftTumors in Mice

FIGS. 25A-B illustrate the combination of voruciclib and venetoclax toimpede growth of DLBCL xenograft tumors. Immune deficient NOD SCID micewere implanted with the RIVA (activated B-cell, or ABC) model of DLBCL.The xenografted tumors were allowed to grow in the NOD SCID host untilthey reached 200 mm³, at which point the xenografted animals wererandomized into 4 study groups: 1. Vehicle control; 2. Voruciclib at 200mpk; 3. Venetoclax at 1 mpk; and 4. Combination of Voruciclib at 200 mpkand Venetoclax at 1 mpk. Voruciclib was dosed by oral gavage daily for 6days of the week with a rest day in between cycles. Venetoclax was dosedby oral gavage on days 1 and 4 of each weekly cycle. Drug effects wereassessed via tumor volume measurements twice weekly by a technicianblinded to the treatment of each subject in the study. N=5-6 animals pertreatment arm.

FIG. 25A graphs the average tumor volume (mm³) for each of the 4 studygroups: vehicle, single agent voruciclib, single agent venetoclax, andthe combination of voruciclib and venetoclax. The single agenttreatments saw a decrease in tumor volume over a longer period of studydays. The combination treatment saw a much higher decrease in tumorvolume size, more than 2-fold decrease of average tumor volume by 3weeks into the study.

FIG. 25B illustrates images of the tumors of animals from each of the 4study groups: vehicle, single agent voruciclib, single agent venetoclax,and the combination of voruciclib and venetoclax. The vehicle tumor sizewas the largest, as outlined by outline 2501. The tumor of single agentvoruciclib is smaller, outlined by outline 2502. The tumor of singleagent venetoclax is smaller than vehicle as well, outlined by outline2503. The combination therapy tumor is outlined by outline 2504, whichis significantly smaller than that of vehicle or either of the singleagent therapies, suggesting a synergistic effect.

FIGS. 26A-B illustrate the combination of voruciclib and venetoclax toimpede growth of DLBCL xenograft tumors. Immune deficient NOD SCID micewere implanted with the U2932 model of DLBCL. The xenografted tumorswere allowed to grow in the NOD SCID host until they reached 200 mm³, atwhich point the xenografted animals were randomized into 4 studygroups: 1. Vehicle control; 2. Voruciclib at 200 mpk; 3. Venetoclax at10 mpk; and 4. Combination of Voruciclib at 200 mpk and Venetoclax at 10mpk. Voruciclib was dosed by oral gavage daily for 6 days of the weekwith a rest day in between cycles. Venetoclax was dosed by oral gavageon days 1 and 4 of each weekly cycle. Drug effects were assessed viatumor volume measurements twice weekly by a technician blinded to thetreatment of each subject in the study. N=5-6 animals per treatment arm.

FIG. 26A graphs the average tumor volume (mm³) for each of the 4 studygroups. The single agent treatments saw a decrease in tumor volume overa longer period of study days. The combination treatment saw almost notumor growth, with the average tumor volume staying consistentthroughout the 29 day study period.

FIG. 26B graphs the average body weight in grams (g) for a subject ineach of the 4 study groups. All 4 study groups showed a stable andconsistent body weight, a general indication of health and safety.

FIGS. 27A-B illustrate the combination of voruciclib and venetoclax toimpede growth of DLBCL xenograft tumors. Immune deficient NOD SCID micewere implanted with the NUDHL1 model of DLBCL. The xenografted tumorswere allowed to grow in the NOD SCID host until they reached 200 mm³, atwhich point the xenografted animals were randomized into 4 studygroups: 1. Vehicle control; 2. Voruciclib at 200 mpk; 3. Venetoclax at50 mpk; and 4. Combination of Voruciclib at 200 mpk and Venetoclax at 50mpk. Voruciclib was dosed by oral gavage daily for 6 days of the weekwith a rest day in between cycles. Venetoclax was dosed by oral gavageon days 1 and 4 of each weekly cycle. Drug effects were assessed viatumor volume measurements twice weekly by a technician blinded to thetreatment of each subject in the study. N=5-6 animals per treatment arm.

FIG. 27A graphs the normalized tumor volume (mm³) for each of the 4study groups. The single agent treatments saw a decrease in tumor volumeover a longer period of study days. The combination treatment saw a muchhigher decrease in tumor volume size, more than 2-fold decrease ofaverage tumor volume by 3 weeks into the study.

FIG. 27B illustrates images of the tumors of animals from each of the 4study groups: vehicle, single agent voruciclib, single agent venetoclax,and the combination of voruciclib and venetoclax. The vehicle tumor sizewas the largest, as outlined by outline 2701. The tumor of single agentvoruciclib is smaller, outlined by outline 2702. The tumor of singleagent venetoclax is smaller than vehicle as well, outlined by outline2703. The combination therapy tumor is outlined by outline 2704, whichis significantly smaller than that of vehicle or either of the singleagent therapies, suggesting a synergistic effect.

FIG. 28 illustrates the combination of voruciclib and venetoclax toimpede growth of DLBCL xenograft tumors. Immune deficient NOD SCID micewere implanted with the SUDHL4 model of DLBCL. The xenografted tumorswere allowed to grow in the NOD SCID host until they reached 200 mm³, atwhich point the xenografted animals were randomized into 4 studygroups: 1. Vehicle control; 2. Voruciclib at 200 mpk; 3. Venetoclax at25 mpk; and 4. Combination of Voruciclib at 200 mpk and Venetoclax at 25mpk. Voruciclib was dosed by oral gavage daily for 6 days of the weekwith a rest day in between cycles. Venetoclax was dosed by oral gavagedaily for 6 days of the week with a rest day in between cycles. Drugeffects were assessed via tumor volume measurements twice weekly by atechnician blinded to the treatment of each subject in the study. N=5-6animals per treatment arm. FIG. 28 graphs the normalized tumor volume(mm³) for each of the 4 study groups. The single agent treatments saw anincrease in tumor volume over the study days. The combination treatmentsaw less tumor growth than the single agent treatments and vehicletreatment.

Each lane of FIG. 29 represents an individual SUDHL4 xenograft.Together, the Western blot illustrates that voruciclib restores p53abrogated by venetoclax. Tumors were harvested on Day 26, 4 hours posttreatment. Each tumor was fragmented into 2. One half was used for FFPEbased IHC analysis, and the other half was used for Western blotanalysis.

Example 13 Voruciclib Suppresses MCL1 Expression in AML Cell Lines

FIGS. 30A-30C illustrate that voruciclib has single agent activity inAML cell lines. AML cell lines (SKM-1, MV-4-11, and OCI-AML-3) wereeither left untreated of were exposed to clinically achievable levels ofvoruciclib (1.5 and 5.0 micromolar) for 2, 6, or 24 h. At the given timepoints, cells were lysed and subjected to western blot analysis withantibodies that specifically recognize phosphorylated RNA pol II(pSer2), MCL-1, cleaved PARP, and Beta-actin. In all three AML celllines (SKM-1, MV-4-11, and OCI-AML-3), Voruciclib suppresses MCL-1 andinduces apoptosis, as shown within outlines 3001, 3003, and 3005.Cleaved PARP (cPARP) may be used as a biomarker of apoptosis. In allthree AML cell lines (SKM-1, MV-4-11, and OCI-AML-3), Voruciclib inducesapoptosis, as shown within outlines 3002, 3004, and 3006.

Example 14 Voruciclib and Venetoclax Combination Induces Cell Death inAML Cell Lines

FIG. 31 illustrates that the combination of voruciclib and venetoclaxinduce synergistic cell death in AML cell lines. AML cell lines (SKM-1,MV-4-11, and OCI-AML-3) were either left untreated of were exposed tolevels of voruciclib (1.5 micromolar), venetoclax (100 nM) that werepreviously shown to be under the levels required to induce single agentapoptosis or the two drugs in combination at these sub-effectiveconcentrations for 24 h. At the given time points, cells were lysed andsubjected to western blot analysis with antibodies that specificallyrecognize phosphorylated RNA pol II (pSer2), MCL-1, cleaved PARP, andBeta-actin. As shown within the outlines 3101 and 3102, the combinationof voruciclib and venetoclax induces apoptosis (PARP), when neitheragent alone induces apoptosis. Venetoclax is observed to induce MCL-1upregulation in all three cell lines. Voruciclib dampens MCL-1 drivenresistance mechanism in all three cell lines.

Example 15 Voruciclib and Venetoclax Combination Impedes Tumor Growth inAML Xenografts

FIG. 32 illustrates that the combination of voruciclib and venetoclaximpedes tumor growth in SKM1 AML xenografts. Single agent therapy ofvenetoclax has a normalized tumor volume similar to that of the vehicle.voruciclib single agent treatment exhibits slightly lower tumor volume,whereas the combination of voruciclib and venetoclax greatly impedestumor growth, with the resulting tumor volume approximately half of thatof the vehicle by day 22 of the study.

Example 16 Voruciclib Induces Apoptosis as a Single Agent in DLBCLModels

FIG. 33 illustrates that voruciclib-induced apoptosis correlates withrepression of MCL-1. Three DLBCL models were used: SUDHL4, RIvA, andU2932. Cleaved PARP (cPARP) may be used as a biomarker of apoptosis. TheWestern blot shows that apoptosis increases as the concentration ofvoruciclib increases. Similarly, voruciclib suppresses MCL-1 and inducesapoptosis as the concentration of voruciclib increases.

While preferred embodiments of the present invention have been shown anddescribed herein, it will be obvious to those skilled in the art thatsuch embodiments are provided by way of example only. Numerousvariations, changes, and substitutions will now occur to those skilledin the art without departing from the invention. It should be understoodthat various alternatives to the embodiments of the invention describedherein may be employed in practicing the invention. It is intended thatthe following claims define the scope of the invention and that methodsand structures within the scope of these claims and their equivalents becovered thereby.

1-24. (canceled)
 25. A method of treating a cancer comprisingadministering to a subject in need thereof a therapeutically effectiveamount of a CDK inhibitor represented by Formula I:

or a pharmaceutically acceptable salt thereof, wherein: R₁ is optionallysubstituted phenyl; R₂ and R₃ are each independently selected fromhydroxy and OR₈, wherein R₈ is optionally substituted C₁-C₁₀-alkyl; R₄is optionally substituted C₁-C₄-alkyl; and R₉ is hydrogen or optionallysubstituted C₁-C₄-alkyl; and a therapeutically effective amount of aproteasome inhibitor.
 26. The method of claim 25, wherein the compoundor salt of Formula I is represented by Formula Ia:


27. The method of claim 25, wherein R₁ is optionally substituted withone or more substituents independently selected from hydroxy, cyano,halo, amino, C₁-C₄-alkyl, C₁-C₄-alkoxy, C₁-C₄-hydroxyalkyl,C₁-C₄-haloalkyl, and nitro.
 28. The method of claim 27, wherein R₁ issubstituted with one or more substituents independently selected fromhalo and C₁-C₄-haloalkyl.
 29. The method of claim 28, wherein R₁ is2-chloro-4-trifluoromethylphenyl.
 30. The method of claim 25, wherein R₂and R₃ are each independently selected from hydroxy and OR₈, wherein R₈is C₁-C₁₀-alkyl optionally substituted with one or more substituentsindependently selected from hydroxy, cyano, halo, amino, ═O, ═S,C₁-C₄-alkoxy, and nitro.
 31. The method of claim 30, wherein R₂ and R₃are each hydroxy.
 32. The method of claim 25, wherein R₄ is C₁-C₄-alkylsubstituted with one or more substituents selected from hydroxy, cyano,halo, amino, ═O, ═S, C₁-C₄-alkoxy, and nitro.
 33. The method of claim32, wherein R₄ is C₁-C₄-alkyl substituted with one or more substituentsselected from hydroxy, cyano, halo, amino, ═O, ═S, C₁-C₄-alkoxy, andnitro.
 34. The method of claim 33, wherein R₄ is 2-hydroxymethyl. 35.The method of claim 25, wherein R₉ is C₁-C₄-alkyl optionally substitutedwith hydroxy, cyano, halo, amino, ═O, ═S, C₁-C₄-alkoxy, and nitro. 36.The method of claim 35, wherein R₉ is methyl.
 37. The method of claim25, wherein the compound of Formula I is represented by formula Ib:

or a pharmaceutically acceptable salt thereof.
 38. The method of claim25, wherein the proteasome inhibitor is selected from bortezomib,marizomib, ixazomib, disulfiram, epigallocatechin-3-gallate,salinosporamide A, carfilzomib, ONX 0912, CEP-18770, MLN9708,epoxomicin, MG132 and a pharmaceutically acceptable salt of any onethereof.
 39. The method of claim 38, wherein the proteasome inhibitor isselected from bortezomib, marizomib, ixazomib, and a pharmaceuticallyacceptable salt of any one thereof.
 40. The method of claim 25, whereinthe cancer is a blood cancer.
 41. The method of claim 40, wherein theblood cancer is selected from acute myeloid leukemia (AML), chronicmyeloid leukemia (CML), acute lymphocytic lymphoma (ALL), and chroniclymphocytic leukemia (CLL), diffuse large B-cell lymphoma (DLBCL),primary mediastinal B-cell lymphoma, intravascular large B-celllymphoma, follicular lymphoma, small lymphocytic lymphomia (SLL), mantlecell lymphoma, marginal zone B-cell lymphomas, extranodal marginal zoneB-cell lymphomas, nodal marginal zone B-cell lymphoma, splenic marginalzone B-cell lymphoma, Burkitt lymphoma, lymphoplasmacytic lymphoma, andprimary central nervous system lymphoma.
 42. The method of claim 41,wherein the blood cancer is diffuse large B-cell lymphoma, acute myeloidleukemia or chronic lymphocytic leukemia.
 43. The method of claim 25,wherein the cancer is triple negative breast cancer (TNBC).
 44. Themethod of claim 25, wherein the CDK inhibitor and the proteasomeinhibitor are administered concurrently.
 45. The method of claim 25,wherein the CDK inhibitor and the proteasome inhibitor are administeredsequentially within about 12 hours of each other.
 46. The method ofclaim 45, wherein the CDK inhibitor and the proteasome inhibitor areadministered sequentially within about 5 hours of each other.
 47. Themethod of claim 25, wherein the CDK inhibitor and the proteasomeinhibitor are co-formulated in a pharmaceutical composition.
 48. Themethod of claim 25, wherein the CDK inhibitor and the proteasomeinhibitor are administered daily, every other day or every third day.49-66. (canceled)